©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Function of the NC1 Domains in Type IV Collagen (*)

(Received for publication, May 31, 1995; and in revised form, July 28, 1995)

Albert Ries (1) Jürgen Engel (2) Ariel Lustig (2) Klaus Kühn (1)(§)

From the  (1)Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany and (2)Biozentrum der Universität Basel, CH-4056 Basel, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha1(IV)NC1 and alpha2(IV)NC1 homodimers but no heterodimers. The hexamers were dissociated at low pH, separated into monomers and dimers, and submitted to reconstitution experiments. Only alpha1(IV)NC1 dimers were able to reconstitute a hexameric complex. alpha1(IV)NC1 and alpha2(IV)NC1 monomers as well as the alpha2(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 alpha(IV) chains before molecule formation.


INTRODUCTION

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 alpha(IV) chains, alpha1(IV) to alpha6(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 [alpha1(IV)](2)alpha2(IV) is ubiquitous to all basement membranes, whereas the [alpha3(IV)](2)alpha4(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 alpha5(IV) and alpha6(IV) chains also form a common molecule is unclear. Both chains are present in several tissues. However, alpha5(IV) is mainly expressed in kidney, whereas the highest expression of alpha6(IV) has been observed in esophagus and lung(14) .

Comparison of the amino acid sequences of alpha(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) .


MATERIALS AND METHODS

Isolation of Hexameric, Dimeric, and Monomeric NC1 Domains

The hexameric NC1 domain was isolated from human placenta similarly to the two-step collagenase procedure described previously (6) . The first collagenase treatment of formic acid-washed tissue (1000 units of type IV clostridium peptidase, Millipore, per kg of wet tissue) was carried out in 30 mM Tris-HCl, pH 7.6, containing 10 mM CaCl(2) at 20 °C for 24 h. The second collagenase treatment (2 units/mg of protein) was performed at 37 °C for 16 h. The digest was stopped with EDTA, dialyzed against 50 mM ammonium carbonate, and lyophilized. For final purification, the protein was dissolved in 1 M CaCl(2), 0.50 mM Tris-HCl, pH 7.4, and passed over an agarose A-1 column.

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 times 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 (^1)and chromatographic separation did not give evidence for the presence of alpha3(IV)-, alpha4(IV)-, alpha5(IV)-, and alpha6(IV)NC1 domains in the hexameric complex isolated from placenta. Also, Western blot analysis with an antiserum against the alpha5(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(2), 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 times 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.

SDS-Polyacrylamide Gel Electrophoresis

Aliquots of the fractions eluted from the Superose 6 column, containing equal amounts of protein, were diluted to 1 ml, precipitated with trichloroacetic acid, dissolved in 4% SDS, 173 mM Tris-HCl, pH 6.8, 10% glycerol, and, after centrifugation, separated at 200 V for 1 h on a 8-18% polyacrylamide gel (90 times 90 cm times 0.5 mm) prepared from a stock solution of 30% acrylamide containing 0.8% bisacrylamide.

Immunological Assays

Antisera against purified alpha1(IV)NC1 and alpha2(IV)NC1 domains separated according to (7) were raised in guinea pigs by two subcutaneous injections of protein (0.3 mg each) together with incomplete Freund's adjuvant. Immunoblotting was performed after electrophoretic separation and transfer to nitrocellulose membranes (Trans-Blot, Bio-Rad) following the manufacturer's instructions (Biometra).

Analytical Ultracentrifugation and Electron Microscopy

Sedimentation velocity and equilibrium experiments were performed with an Optima XL-A analytical ultracentrifuge (Beckman Instruments) at 20 °C in 12-mm Epon double-sector cells in an AnD rotor. Velocity experiments were performed at a rotor speed of 56,000 rpm. Proportions of differently sedimenting materials were estimated from the relative heights of peaks in the sedimentation profiles. For equilibrium experiments, absorbance A was measured at 280 nm, and the molar mass was determined from lnA versus r^2 plots where r is the distance from the rotor center. Rotor speeds between 9,000 rpm and 22,000 rpm were selected for best resolution. In cases where there was only little contamination of the major species, it was possible to estimate the individual molar masses, but in other cases weight average molar masses were determined in short column runs according to the method of Van Holde and Baldwin(15) . An average partial specific volume of v = 0.73 ml/g was used, which was based on the amino acid composition of NC1 hexamers.

Electron microscopy of the NC1 domains after rotary shadowing was performed as described previously(16) .


RESULTS

Separation of Monomeric and Dimeric NC1

The hexameric NC1 complex, isolated from human placenta with the two-step collagenase procedure, was submitted to SDS-polyacrylamide gel electrophoresis under nonreducing conditions (Fig. 1). Under the special conditions used, 200 V for 1 h, the NC1 dimers were resolved into four bands. Western blot analysis, using alpha1(IV)NC1- and alpha2(IV)NC1-specific antisera, revealed that the two upper bands consist of alpha1(IV)NC1 dimers, whereas the two bands with higher electrophoretic mobility represent alpha2(IV)NC1 dimers (Fig. 1). Hybrid dimers between alpha1(IV)NC1 and alpha2(IV)NC1 were not observed.


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 alpha1 dimers, alpha2 dimers, and alpha1(IV)NC1 and alpha2(IV)NC1 are designated. 2, Western blot with an antiserum against alpha1(IV)NC1. 3, Western blot with an antiserum against alpha2(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, alpha1(IV)NC1 eluted earlier than the alpha2(IV)NC1 domains. There are fractions which contain only alpha1(IV)NC1 dimers and others with mainly alpha2(IV)NC1 dimers. Densitometric analysis of gels from seven different preparations revealed that 79% of the alpha1(IV)NC1 and 54% of the alpha2(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 alpha1(IV) chains within a collagen IV monomer via the alpha1(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 alpha1(IV)NC1 and alpha2(IV)NC1 dimers and monomers are indicated.



Reconstitution Experiments

These experiments were carried out to test the affinity of monomeric and dimeric NC1 domains for each other at neutral pH and their ability to form ordered hexameric and trimeric complexes. Native hexamers, alpha1(IV)NC1 dimers, alpha2(IV)NC1 dimers, and a mixture of alpha1(IV)NC1 and alpha2(IV)NC1 monomers in formate buffer, of pH 3, were dialyzed for 16 h against a phosphate buffer, pH 7.4, and finally chromatographed on a Superose 6 column to analyze for reformed complexes and the presence of nonassociated monomers and dimers (Fig. 3).


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, alpha1(IV)NC1 dimers. C, alpha2(IV)NC1 dimers with minor content of alpha1(IV)NC1 dimers, arrows designate alpha1(IV) and alpha2(IV) dimers. Of the alpha1(IV) dimers, only the fraction with the higher electrophoretic mobility is present. D, alpha1(IV)NC1 dimers as in B to which a mixture of alpha1(IV)NC1 and alpha2(IV)NC1 monomers has been added. E, alpha1(IV)NC1 and alpha2(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 alpha1(IV)NC1 dimer was able to reform a hexameric complex (Fig. 3B). The hexameric fraction observed after reconstitution of alpha2(IV)NC1 dimers is due to a small amount of alpha1(IV)NC1 dimers still present in the preparation (Fig. 3C). The alpha1(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 alpha1(IV)NC1 dimers in the presence of alpha1(IV)NC1 and alpha2(IV)NC1 contained monomers, and it is notable that alpha2(IV)NC1 was preferentially incorporated. A mixture of alpha1(IV)NC1 and alpha2(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 alpha1 dimers, 80% recovery of intact hexamers was observed. The alpha2(IV) dimer fraction showed 30% hexamers. This is apparently due to contamination with alpha1(IV) dimers (see Fig. 3C).

The hexameric complexes reconstituted from a native NC1 mixture or from isolated alpha1(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 alpha1(IV)NC1 dimers.




DISCUSSION

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 alpha1(IV)NC1 and alpha2(IV)NC1 homodimers but no alpha1-alpha2 heterodimers have been found. Striking is the occurrence of two species for the alpha1(IV)NC1 as well as for the alpha2(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 alpha1(IV)NC1 dimers were able to reform a hexameric complex, which could not be differentiated from the native complex in the electron microscope. The alpha1(IV)NC1 and alpha2(IV)NC1 monomers, as well as the alpha2(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 alpha1(IV)NC1 domains is accompanied by a drastic alteration of their aggregation behavior. In the presence of a mixture of alpha1(IV)NC1 and alpha2(IV)NC1 monomers, mainly the latter were incorporated into the hexamers formed by alpha1(IV)NC1 dimers, indicating a tendency to restore the native ratio of alpha1(IV) to alpha2(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 alpha1(IV) and one alpha2(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 alpha1(IV) and alpha2(IV) chains in a molecule is also unknown, and further work is needed to elucidate the specificity of chain recognition.


FOOTNOTES

*
This work was supported by Swiss National Science Foundation Grant 31-32251.91 (to J. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Max-Planck-Institut für Biochemie, D-82152 Martinsried b. München, Germany. Tel.: 49-089-8578-2423; Fax: 49-089-8578-2422.

(^1)
The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of Hanna Wiedemann.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.