From the Department of Biochemistry and Molecular
Biology, University of Kansas Medical Center, Kansas City, Kansas
66160-7421, the § Division of Immunology, Shigei Medical
Research Institute, Okayama 701-02, Japan, and the ¶ Department of
Molecular Biology and Biochemistry, Okayama University Medical School,
Okayama 700, Japan
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ABSTRACT |
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Glomerular basement membrane (GBM) plays a
crucial function in the ultrafiltration of blood plasma by the kidney.
This function is impaired in Alport syndrome, a hereditary disorder
that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the
present study, the supramolecular organization of type IV collagen of
GBM was investigated. This was accomplished by using pseudolysin (EC
3.4.24.26) digestion to excise truncated triple-helical protomers for
structural studies. Two distinct sets of truncated protomers were
solubilized, one at 4 °C and the other at 25 °C, and their chain
composition was determined by use of monoclonal antibodies. The 4 °C
protomers comprise the 1(IV) and
2(IV) chains, whereas the
25 °C protomers comprised mainly
3(IV),
4(IV), and
5(IV)
chains along with some
1(IV) and
2(IV) chains. The structure of
the 25 °C protomers was examined by electron microscopy and was
found to be characterized by a network containing loops and supercoiled
triple helices, which are stabilized by disulfide cross-links between
3(IV),
4(IV), and
5(IV) chains. These results establish a
conceptual framework to explain several features of the GBM
abnormalities of Alport syndrome. In particular, the
3(IV)·
4(IV)·
5(IV) network, involving a covalent linkage
between these chains, suggests a molecular basis for the conundrum in
which mutations in the gene encoding the
5(IV) chain cause defective
assembly of not only
5(IV) chain but also the
3(IV) and
4(IV)
chains in the GBM of patients with Alport syndrome.
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INTRODUCTION |
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Basement membranes are specialized extracellular matrices that compartmentalize tissues, provide substrata for cells, and provide signals for differentiation, maintenance and remodeling of tissues. Prominent basement membranes are the lens capsule (LBM)1 of eye, seminiferous tubule basement membrane (STBM) of testis, and glomerular basement membrane (GBM) of kidney glomerulus. GBM plays a crucial function in the normal purification of blood plasma, and its function is impaired in at least three prominent renal diseases: diabetic nephropathy, Goodpasture syndrome, and Alport syndrome. In Goodpasture syndrome, a rapidly progressive autoimmune disorder, the target autoantigen is type IV collagen protein, whereas in Alport syndrome, a hereditary form of progressive renal disease, the underlying molecular defects are mutations in the genes encoding type IV collagen (1).
Type IV collagen, the major component of mammalian basement membranes,
is a family of six (IV) chains (1). These are designated
1(IV) to
6(IV) and they are encoded by six distinct genes, COL4A1 to COL4A6. The chains are distributed among various basement
membranes in a tissue-specific manner, wherein the
1(IV) and
2(IV) chains have a ubiquitous distribution and the
3(IV) to
6(IV) chains have a restricted distribution (2-5). For example, LBM
is composed mainly of the
1(IV) and
2(IV) chains (6) and STBM is
composed of all six
(IV) chains (7), whereas GBM is composed of five chains,
1(IV) to
5(IV) (5, 6, 8). The chains are assembled into
triple-helical molecules (protomers, Fig. 1) composed of three
(IV)
chains that self-associate to form supramolecular networks. Several
isoforms of protomers are possible that differ in kind and
stoichiometry of chains (1), but only two have been identified, an
[
1(IV)]2
2(IV) (9) and an [
3(IV)]2
4(IV) protomer (7, 10). Correspondingly, several kinds of
supramolecular networks are also possible that differ with respect to
isoform composition (1), as was proposed upon the discovery of the
3(IV) and
4(IV) chains (11). Three such networks have been identified: a ubiquitous
1(IV)·
2(IV) network and both an
1(IV)-
6(IV) and an
3(IV)-
6(IV) network in STBM (7). An
3(IV)-containing network, that does not contain
1(IV) and
2(IV) chains, has been identified in GBM (10, 12).
In Alport syndrome, mutations occur in the COL4A3,
COL4A4, and COL4A5 genes encoding the 3(IV),
4(IV), and
5(IV) chains. About 80% of the affected families
exhibit X-linked inheritance of mutations in the COL4A5 gene
(13, 14), while some of the remainder inherit autosomal recessive
mutations in
3(IV) and
4(IV) chains (15, 16). To date, over 200 mutations have been found in the COL4A5 gene (13, 14, 17,
18). However, the molecular mechanisms by which gene mutations in type
IV collagen cause defects in GBM are unknown. The abnormal GBM in most
patients with X-linked Alport syndrome contains only the
1(IV) and
2(IV) chains and is devoid of the
3(IV),
4(IV), and
5(IV)
chains (5, 8, 19-21). A recent study has revealed that this phenomenon reflects an arrest of an early developmental switch, wherein the
1(IV) and
2(IV) chains persist and are not replaced by the
3(IV),
4(IV), and
5(IV) chains that are necessary to form a
mature GBM (22). How mutations in the
5(IV) chain cause defective assembly of not only the
5(IV) chain, but also the
3(IV) and
4(IV) chains, remains a conundrum. The mechanism linking the assembly of these three chains could be at the protein level in which
the
3(IV),
4(IV), and
5(IV) chains are structurally linked into a supramolecular network (5, 22), either at the level of
triple-helical protomers or at the linkages between triple-helical protomers. The linkage mechanism may also involve the
transcriptional/translational level, because the mRNA expression of
the
3(IV),
4(IV), and
5(IV) chains appears to be coordinated
(22).
The purpose of the present study was to characterize the supramolecular
organization of type IV collagen chains in GBM. This was accomplished
using pseudolysin cleavage to excise soluble triple-helical molecules
for determination of their chain organization. The results revealed
that the 3(IV),
4(IV), and
5(IV) chains exist in a novel
supramolecular network that is cross-linked by disulfide bonds between
triple helices. Moreover, the findings establish a structural linkage
between the
3(IV),
4(IV), and
5(IV) chains in GBM, suggesting
a molecular basis for the conundrum in which COL4A5 gene
mutations in Alport syndrome cause defective assembly of not only the
5(IV) chain, but also the
3(IV) and
4(IV) chains.
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EXPERIMENTAL PROCEDURES |
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Materials-- Bovine kidneys were collected from a local slaughterhouse. GBM was prepared as described by Freytag et al. (23). Pseudolysin was from Nagase Biochemical, Fukuchiyama, Japan; bacterial collagenase (CLSPA) from Worthington; Reacti-gel and bicinchoninic acid-protein assay reagents from Pierce; Sephacryl S-300, S-400, S-1000, and CNBr-activated Sepharose 4B from Amersham Pharmacia Biotech; a C18 reversed-phase HPLC column (201 TP 104, 10 µm) from Vydac; nitrocellulose from Schleicher & Schuell; Immobilon from Millipore; a Bio-Gel TSK 250 HPLC gel-filtration column (600 mm length) from Bio-Rad; and prestained protein standards from Life Technologies, Inc.
Preparation of 4 °C Truncated Protomers from Bovine
GBM--
One gram of GBM was suspended in 100 ml of 150 mM
NaCl, 50 mM Tris-HCl, 2 mM CaCl2,
pH 7.5. To this, 10 mg of pseudolysin was added and digestion was
carried out at 4 °C for 36 h. The suspension was then
centrifuged at 10,000 rpm (16,000 × g) in a Sorvall
HB-4 rotor to remove un-solubilized GBM. The supernatant contained most
of the 1(IV) and
2(IV) chains and the pellet contained primarily
3(IV),
4(IV), and
5(IV) chains. The supernatant solution was
brought to 20 mM EDTA, and the digested collagen IV was
precipitated by increasing the NaCl concentration to 2 M
(23). After 4 h, the precipitate was sedimented at 10,000 rpm
(16,000 × g) in a Sorvall HB-4 rotor for 25 min. The
pellet was dissolved in 75 ml of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. The residual pseudolysin was removed
by filtration through a Sephacryl S-1000 column that had been
equilibrated against 150 mM NaCl, 50 mM
Tris-HCl, pH 7.5. The fractions free of pseudolysin were pooled and
used for further experiments. The 4 °C protomers accounted for 20%
of the weight of GBM, based upon absorbance measurements at 280 nm of
Sephacryl S-1000 fractions, using an absorbance of 0.38 for a 1 mg/ml
in a cell of 1 cm pathlength. The value 0.38 was calculated from the
amino acid composition of type IV collagen (24). GBM contains ~40%
by weight of type IV collagen (25); thus, the 4 °C protomers
comprise ~50% of the type IV collagen of GBM.
Preparation of 25 °C Truncated Protomers from Bovine
GBM--
The pellet from the 4 °C pseudolysin digestion was washed
three times with cold water, once with 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and then suspended in 50 ml of the
same buffer. The suspension was made 0.5% in pseudolysin and 2 mM in CaCl2, and digestion was carried for
24 h at room temperature (25 °C). The reaction was arrested by
making the digest 20 mM in EDTA. The suspension was
centrifuged for 25 min at 10,000 rpm (16,000 × g).
Collagen IV fragments were precipitated by adding NaCl to the cold
(4 °C) supernatant solution (final concentration 2 M).
After 4 h the precipitate was collected by centrifugation. The
pellet was dissolved in 150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, and the collagen IV fragments precipitated once again
with NaCl and centrifuged as described above. The pellet was suspended
in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA and filtered through Sephacryl S-1000 to remove residual pseudolysin. The fractions free of pseudolysin were pooled and
used for subsequent experiments. The solubilized 25 °C protomers accounted for 18% of the weight of GBM, and ~45% of the type IV collagen of GBM, based upon absorbance measurements at 280 nm of
Sephacryl S-1000 fractions, using an absorbance of 0.38 for a 1 mg/ml
in a cell of 1 cm pathlength. These protomers required pseudolysin at
25 °C for solubilization, as they remained insoluble after
increasing the temperature to 25 °C in the absence of pseudolysin. The protein that remained insoluble even after the 25 °C pseudolysin digestion represented 5% of the total GBM, as determined by dry weight
measurements, and had a type IV -chain composition similar to that
of the 25 °C protomers.
Fractionation of 25 °C Truncated Protomers under Denaturing Conditions-- Truncated protomers obtained by 25 °C pseudolysin digestion of GBM and fractionated through the Sephacryl S-1000 column were dialyzed against 6 M guanidine-HCl for 36-48 h and then fractionated on a Sephacryl S-400 column that was equilibrated in the same solution. The fractions were pooled, concentrated using Amicon (Mr cut-off, 30,000) and used for further experiments.
Analysis of NC1 Domains from 4 °C and 25 °C Truncated
Protomers--
The NC1 domains were released from truncated protomers
by digestion with bacterial collagenase, and the NC1 hexamers were purified on either a Bio-Sil TSK-250 HPLC gel filtration column or a
Sephacryl S-300 column (1.6 × 50 cm) as described previously (26,
27). Fifty mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl and 0.05% NaN3, was used for
equilibration and elution of the column. The NC1 hexamers were then
analyzed by HPLC to quantitate the NC1 domains and by two-dimensional
electrophoresis and SDS-PAGE to identify their (IV) origins using
previously established techniques (7).
Reduction and Alkylation of 25 °C Truncated Protomers under Native Conditions-- A sample from the pseudolysin digest that had been fractionated by filtration through a Sephacryl S-1000 column was dissolved in 0.5 M NaCl, 0.1 M Tris-HCl, 0.5% 2-mercaptoethanol (v/v), pH 8.3, at approximately 1 mg/ml protein concentration. The mixture was stirred for 24 h at room temperature. A 2-3-fold molar excess of sodium iodoacetate was then added, and the mixture was stirred for 4 h in the dark (28). The mixture was then centrifuged, and the supernatant was used for further experiments.
Pepsin Digestion of 25 °C Truncated Protomers-- Pepsin digestion was performed as described by Miller and Rhodes (29). The pooled 25 °C protomers from the Sephacryl S-1000 column were precipitated by making the solution 2 M in NaCl. The precipitate was collected by centrifugation, dissolved in 0.5 M acetic acid (5 mg of protein/ml and then dialyzed against 0.5 M acetic acid at 4 °C. Pepsin was added at 1:10 pepsin-to-substrate ratio, and digestion was performed at 4 °C for 24 h. The collagen IV products were purified by two precipitations with 2 M NaCl, 0.5 M acetic acid. The second precipitate was solubilized in 50 mM Tris-HCl, pH 7.5, dialyzed against 50 mM Tris-HCl, pH 7.5, and stored at 4 °C until further use.
Electrophoretic Techniques-- Two-dimensional electrophoresis was performed as described previously but using 6-22% gradient gels in the second dimension (27). In some cases, either 4-22% or 5-15% gels were used for hexamer or large fragments, respectively. The approximate sizes of the fragments were determined as described previously (30). To determine approximate sizes of complex protomeric molecules, 1.5% agarose gel electrophoresis was performed as described previously (30) but using slab gels instead of cylindrical gels. Ascaris suum basement membrane collagen (31), calf skin collagen, and rabbit skeletal muscle myosin (30) were used as molecular weight standards.
Immunochemical Techniques--
To prepare Western blots, the
electrophoretically separated proteins were transferred to
nitrocellulose, blocked with 2% bovine serum albumin, reacted with
primary antibody, and stained with nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate substrates (32). Alkaline
phosphatase conjugate was used as secondary antibody. Primary
monoclonal antibodies directed against human 1(IV)-
6(IV)NC1
domains were used for Western blots. The specificity of the monoclonal
antibodies was previously established for both human and bovine NC1
domains (8, 33) and they were used to determine the tissue distribution
of
(IV) chains. About 5 µg of protein was loaded in each lane in
SDS-PAGE and 10 µg for two-dimensional electrophoresis.
Two-dimensional gels were stained with silver as described by Morrissey
(34).
Chemical Analysis-- Protein and hydroxyproline concentrations were determined by published procedures (35, 36).
Electron Microscopy-- Rotary-shadowing electron microscopy studies were performed on 4 °C and 25 °C truncated protomers using conditions (26) adapted from Shotton et al. (37). Samples (20 µg/ml), in 25% glycerol containing either 150 mM ammonium bicarbonate or 50 mM acetic acid, were sprayed onto freshly cleaved mica sheets, and were rotary-shadowed with platinum at 9° followed by carbon at 90°. Replicas were examined with a JEOL JEM-100CX II electron microscope. The contour length of molecules was measured with a Summagraphics Data Tablet (model MM1201).
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RESULTS |
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Solubilization of Truncated Protomers of Type IV Collagen by Pseudolysin Digestion of GBM
In our previous studies of STBM and LBM (7, 38), digestion with
pseudolysin at 4 °C was established as a procedure to solubilize
truncated protomers of type IV collagen (Fig.
1) that retain a portion of the
triple-helical domain and the complete NC1 carboxyl domain. Such
truncated molecules can provide information about the chain composition
of protomers and the organization and linkages of protomers in
supramolecular networks. The retention of the NC1 domains on the
truncated chains allows for the identification of the kind (1 to
6) of
(IV) chain using NC1-specific monoclonal antibodies. In the
present study, truncated protomers were obtained under two separate
conditions of pseudolysin digestion. First, the digestion was performed
at 4 °C, which solubilized a set of truncated protomers, and then a
second digestion of the residual insoluble fraction was performed at
25 °C, which solubilized another set of truncated protomers. The two
populations of truncated protomers, designated 4 °C and 25 °C
protomers, were characterized (see below) with respect to chain
composition and supramolecular organization. The chain compositions
were determined using the same chain-specific monoclonal antibodies
that were used to determine the distribution of chains in renal tissue
(8, 33).
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Gel Filtration Analysis-- The elution profiles of the digestion mixtures of truncated protomers that were filtered through Sephacryl S-1000 are shown in Fig. 2. For both 4 °C and 25 °C protomers, the elution profiles are broad. However, in each case, SDS-PAGE analysis (data not shown) revealed that most of the fractions contained fragments of the same sizes upon reduction of disulfide bonds, indicating a large heterogeneity in size of the constituents of the disulfide-linked complexes. The major result of the fractionation was the removal of pseudolysin. Fractions 20-120 were recombined, and the 4 °C and 25 °C protomers were analyzed by SDS-PAGE to determine the size and distribution of fragments that were dissociated by SDS (Fig. 2, inset). In the 4 °C protomers, some fragments were retained in the stacking gel and those that were resolved by the gel exhibited apparent Mr values of <16,000 to >200,000. The apparent Mr of the most intense fragment was 160,000. In contrast, those of the 25 °C protomers were of larger size, with a major fragment with an apparent Mr >200,000.
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Chain Composition of 4 °C and 25 °C Protomers--
The
identity of (IV) chains comprising these molecules was determined by
analysis of their respective NC1 domains that were released upon
collagenase digestion. The identity of the NC1 domains was determined
by SDS-PAGE analysis and Western blotting using chain-specific
monoclonal antibodies (Fig. 3). The NC1
domains of the 4 °C protomers showed reactivity with anti-
1(IV)
and
2(IV) antibodies, with no reactivity to antibodies directed
against
3(IV),
4(IV),
5(IV), and
6(IV) chains (Fig.
3A). These results indicated that pseudolysin cleaved and
selectively solubilized the
1(IV) and
2 chains of GBM at 4 °C.
The NC1 domains of the 25 °C protomers showed reactivity to
antibodies directed against
1(IV),
2(IV),
3(IV),
4(IV), and
5(IV) chains, and no reactivity toward the
6(IV) chain (Fig.
3B). This indicated that the 25 °C protomers were mainly
composed of the
3(IV),
4(IV), and
5(IV) chains. Goodpasture
(GP) autoantibodies and Alport (ALP) alloantibodies also reacted with
the NC1 domains of the 25 °C protomers, indicating the presence of
the
3(IV) NC1 domain (1, 7).
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Supramolecular Organization of 4 °C and 25 °C Protomers
Rotary Shadowing Electron Microscopy--
The 4 °C truncated
protomers, comprising the 1 and
2(IV) chains, were analyzed by
rotary shadowing electron microscopy (Fig. 5). In Fig. 5A, they are shown
to have a triple-helical (rodlike) segment, 300 ± 28 nm in
length, linked to a globular NC1 domain and are dimerized through
NC1-NC1 interactions, forming molecules with lengths of about 600 nm.
These molecules have a very similar length to the
1(IV)·
2(IV)
protomers (triple-helical domain = 287 nm) that were solubilized
from STBM by pseudolysin at 4 °C (7), but they have a length twice
that of the
1(IV)·
2(IV) protomers solubilized from LBM (38).
About 5-10% of the GBM protomers were not connected by NC1-NC1
interactions and thus existed as monomers (data not shown). A small
percentage had triple helices of contour length of only 140 nm and
these occurred as dimers (Fig. 5B) and monomers (data not
shown). A small percentage existed as dimers in which one triple-helix
had a length of 300 nm and the other 140 nm (Fig. 5C).
Fragments that originated from the 7 S domain were also observed (Fig.
5D).
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Identification of a Disulfide-linked Complex of the 3(IV),
4(IV), and
5(IV) Chains--
The 25 °C protomers were further
characterized by SDS-agarose gel electrophoresis (Fig.
8A) to estimate the size
distribution of dissociated components with molecular weights
>200,000. The components ranged in apparent molecular weight from
~300,000 to >1,000,000 (Fig. 8A, lane e).
Reduction and alkylation under non-denaturing conditions
(i.e. in 0.5 M NaCl, 0.1 M Tris-HCl,
pH 8.3) produced a component with a Mr 500,000 (Fig. 8A, lane f) indicating the presence of
disulfide cross-links. This Mr 500,000 component
dissociated into multiple polypeptides (Mr
<160,000) upon complete reduction under denaturing conditions (see
below), indicating that it is cross-linked by disulfide bonds. This
Mr 500,000 component was then treated with
pepsin, which degrades the NC1 domains and cleaves at the
noncollagenous interruptions of the collagenous domain. The peptic
product retained an apparent Mr 500,000 (Fig.
8A, lane g), which indicates that the disulfide
cross-links were within the collagenous domain, as those within the NC1
domain would have been removed by the digestion.
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DISCUSSION |
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Pseudolysin digestion of GBM at 4 °C and then at 25 °C
solubilized two distinct sets of truncated protomers of type IV
collagen. Those solubilized at 4 °C were mainly comprising 1(IV)
and
2(IV) chains. By electron microscopy, they exhibited structural
characteristics of
1(IV)·
2(IV) protomers that have been
described for STBM and LBM (7, 38). These include a long triple-helical
segment that connects to the NC1 domain at the carboxyl terminus, and NC1-NC1 interactions, forming dimers of protomers. The protomer length
is about two-thirds of the 450 nm length of the intact molecule (9),
which reflects removal of a large region of the triple helix that
includes the 7 S amino-terminal tetramerization domain. These truncated
molecules, therefore, reflect the existence of a supramolecular network
comprising the
1(IV) and
2(IV) chains in GBM. The existence of
such a network is also supported by the finding that NC1 hexamers
comprising
1(IV) and
2(IV) NC1 domains can be isolated from GBM
(10, 12), although the cited studies could not rule out the presence of
5(IV) and
6(IV) chains.
In contrast to the truncated protomers solubilized at 4 °C, those
solubilized at 25 °C were mainly comprising the 3(IV),
4(IV),
and
5(IV) chains, along with a small amount of
1(IV) and
2(IV)
chains. These exhibited some structural features that are in common
with the
1(IV)·
2(IV) protomers and others that are distinctly
different. The common features include a long triple-helical domain, a
globular NC1 domain and NC1-NC1 interactions causing end-to-end
association of protomers. Distinct differences are evident at the
supramolecular level in which the
3(IV)·
4(IV)·
5(IV) protomers are characterized by loops and supercoiling of the triple helices (Fig. 6A) that are stabilized by inter-protomer
disulfide bonds. At the chain level, the
3(IV),
4(IV), and
5(IV) chains of the complex are covalently linked by disulfide
bonds, exclusive of
1(IV) and
2(IV) chains. Evidence for
disulfide bonds is that the high molecular weight complex does not
dissociate in 6 M guanidine-HCl or 1% SDS in the absence
of a disulfide reducing agent, but in the presence of a reducing agent
it dissociates into its constituent
3(IV),
4(IV), and
5(IV)
chains. Fragments of the
1(IV) and
2(IV) chains could be
separated from the
3(IV)·
4(IV)·
5(IV) complex by exposure
to 6 M guanidine-HCl, indicating that they were not linked
in the complex by disulfide bonds. The 25 °C protomers, therefore,
reflect the existence of a novel supramolecular network in GBM that
comprises the
3(IV),
4(IV), and
5(IV) chains and which is
cross-linked by disulfide bonds between triple helices. Such a network
is also supported by the finding that NC1 hexamers, comprising the
3(IV) and
4(IV) NC1 domains, can be isolated from GBM (10),
although the studies did not address the presence of the
5(IV)
and
6(IV) chains. Moreover, an
3(IV)·
4(IV)·
5(IV) network is consistent with immunocytochemical results showing that the
3(IV),
4(IV), and
5(IV) chains, but not the
6(IV) chain,
are present in GBM (8, 33). A similar network that also contains the
6(IV) chain is present in STBM (7).
A third network comprising 1(IV)-
5(IV) chains may also exist.
The 25 °C protomers contain some
1(IV) and
2(IV) fragments that could be noncovalently associated with the
3(IV),
4(IV), and
5(IV) chains. Such a network is supported by the observation that
NC1 hexamers can also be isolated from GBM that contain the NC1 domains
of
1(IV) and
2(IV) chains as well as
3(IV) and
4(IV) chains
(10). Moreover, the residual insoluble GBM from 25 °C pseudolysin
digestion contains
1(IV)-
5(IV) chains, which could comprise an
1(IV)-
5(IV) network. Such a network that also contains the
6(IV) chain exists in STBM (7). All three networks
1(IV)·
2(IV),
3(IV)·
4(IV)·
5(IV), and
1(IV)-
5(IV) could be connected into one supra-complex through
association of 7 S domains, i.e. the well known
tetramerization of the amino-terminal domain (9), thus forming a single
network. In this case, they would represent regions of that network
within GBM.
The presence of disulfide cross-links between the triple helices
of the 3(IV)·
4(IV)·
5(IV) truncated protomers is a
distinguishing feature not found in the
1(IV)·
2(IV) truncated
protomers. This can be rationalized by the presence of several cysteine
residues in the collagenous domain of the
3(IV) and
4(IV) chains
that are not found in the
1(IV) and
2(IV) chains. Comparison of
the amino acid sequences of the six
(IV) chains (39-48) reveals the following. First, there are 18 conserved cysteine residues among all
six human
(IV) chains: 4 in the 7 S domain, 12 in the NC1 domain,
and 2 in triple-helical interruption IX (Fig.
9). Second, the
3(IV) chain contains
four cysteine residues and the
4(IV) chain contains nine cysteine
residues within the collagenous domain that are not found in the other
four
(IV) chains (Fig. 9). Third, the truncated
3(IV) and
4(IV) chains that are constituents of the
3(IV)·
4(IV)·
5(IV) complex, fraction I (Fig.
6B), have a sufficient length (>1300) residues to contain
these additional cysteine residues, indicating their participation in
the inter-protomer disulfide cross-links that stabilize the
3(IV)·
4(IV)·
5(IV) complex shown in Fig. 7A.
Fourth, the truncated
5(IV)chain of the
3(IV)·
4(IV)·
5(IV) complex has a length of ~1350 residues and contains an NC1 domain. This length is sufficient to contain a
cysteine residue in helical interruption VIII that could participate in
a disulfide cross-link with an
3(IV) or
4(IV) chain, either within a protomer or between protomers. The
5(IV) chain is also linked to the
3(IV) or
4(IV) chain through disulfide bonds
between NC1 domains because the 1100-residue fragment of the
3(IV)·
4(IV)·
5(IV) complex (Fig. 8C) is not of
sufficient length to contain a cysteine residue within the collagenous
domain. Presumably, the disulfide cross-links within the collagenous
domain stabilize the looping and supercoiling of the triple helices to
confer a specialized function to the
3(IV)·
4(IV)·
5(IV)
network.
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The 3(IV)·
4(IV)·
5(IV) supramolecular structure (Fig.
6A) is similar to structures found in vitro and
in situ by Yurchenco and co-workers (49-53) in membranes
known to contain
1(IV) and
2(IV) chains but which were studied
before it was possible to detect
3(IV),
4(IV), and
5(IV). In
one of these studies (49), it was shown that mouse type IV collagen
protomers from the Engelbreth-Holm-Swarm tumor, containing only
1(IV) and
2(IV) chains, could be induced to form a polygonal
network (49). The
3(IV)·
4(IV)·
5(IV) supramolecular structure that we observed is stabilized by disulfide cross-links between the constituent
(IV) chains. Conceivably, the
1(IV)·
2(IV) protomers we observed may have had a similar
suprastructure in vivo, but it was not preserved because of
the absence of disulfide bonds to stabilize it for viewing by electron
microscopy. In addition, our electron microscopy studies were done at
much lower concentrations than those needed to form polygonal networks
in vitro (49).
The finding of an 1(IV)·
2(IV) network and an
3(IV)·
4(IV)·
5(IV) network provides a conceptual
framework to explain several features of the GBM abnormality in Alport
syndrome. First, recent studies reveal that normal glomerular
development involves a developmental switch in which the early
expression of the
1(IV) and
2(IV) chains is replaced by the
expression of the
3(IV),
4(IV), and
5(IV) chains to form a
mature GBM (20, 54). In X-linked Alport syndrome, the switch is
arrested, causing the persistence of the
1(IV) and
2(IV) chains
and the absence of the
3(IV),
4(IV), and
5(IV) chains (20).
Based upon the present results, the switch can now be defined at the
supramolecular level in which the
1(IV)·
2(IV) network is
replaced by the
3(IV)·
4(IV)·
5(IV) network in normal
glomerular development, but in Alport syndrome the switch is arrested.
Second, it is well established that mutations in the
5(IV) chain
cause defective assembly of not only the
5(IV) chain, but the
3(IV) and
4(IV) chains as well (5, 8, 19-21). This conundrum
indicates that the developmental switch contains a mechanism that links
the assembly of all three
(IV) chains. The finding of an
3(IV)·
4(IV)·
5(IV) network, in which all three chains are
covalently linked by disulfide cross-links, provides a possible linkage
mechanism, wherein the
5(IV) chain is required for the assembly of
the
3(IV) and
4(IV) chains. The dependence of one chain on the
assembly of another chain is a well established mechanism for type I
collagen in patients with osteogenesis imperfecta in which mutations in
the
1(I) chain cause defective assembly of the
2(I) chain (55).
The
5(IV) chain requirement could be at: (a) the protomer
level in which an
5(IV) chain together with an
3(IV) and
4(IV)
chain forms a triple-helical protomer or (b) the
supramolecular level in which an
5(IV)-containing protomer is
required for the incorporation of an
3(IV) or
4(IV) containing
protomer. The linkage mechanism may also involve the transcriptional/translational level because the mRNA expression of
the
3(IV),
4(IV), and
5(IV) chains appears to be coordinated (22). The protein assembly and the transcriptional/translational mechanism need not be mutually exclusive because the incorporation of
an
5(IV) chain into extracellular matrix could be required to
modulate the transcription of the
3(IV) and
4(IV) chains.
The 3(IV)·
4(IV)·
5(IV) network also provides an explanation
for the GBM abnormality in a mouse model of autosomal Alport syndrome.
In the collagen COL4A3 knockout mouse, the
3(IV),
4(IV), and
5(IV) chains are absent in the GBM (56, 57). Thus, the
3(IV) chain is crucial for the assembly of the
4(IV) and
5(IV) into an
3(IV)·
4(IV)·
5(IV) network. Potentially, mutations
in the human COL4A3 and COL4A4 genes in recessive
Alport syndrome (15, 16) and mutations in the COL4A4 gene in
benign familial hematuria (58) also cause loss of the
3(IV)·
4(IV)·
5(IV) network.
The fundamental importance of the 3(IV)·
4(IV)·
5(IV)
network for normal glomerular ultrafiltration function is evident from the gene mutations that cause renal failure in Alport syndrome and from
the
3(IV) knockout mouse models (18, 57). The importance is
underscored by the restricted distribution of this network to
peripheral GBM within the nephron. Conceivably, the looping and
supercoiling of the triple helices and the disulfide cross-linking of
the network (Fig. 6A) confer long term stability to the GBM by protecting against proteolytic degradation (20). An increased susceptibility of the
1(IV)·
2(IV) network to the action of
proteases is supported by the observation that: (a) the
level of 3-hydroxyproline, a constituent of type IV collagen, is
elevated in the urine of 20 Alport patients in comparison to that of
the other renal disorders (64), and (b) Alport renal
basement membranes appears more susceptible to proteolysis than normal
GBM (20). Moreover, the
1(IV)·
2(IV) network is more easily
excised from GBM by pseudolysin than the
3(IV)·
4(IV)·
5(IV)
network as described herein, which further supports the hypothesis that
the
3(IV)·
4(IV)·
5(IV) network may protect against
proteolysis.
The present findings also establish the supramolecular structure of the
target molecules in GBM that bind the pathogenic anti-GBM antibodies in
patients with Goodpasture syndrome and in patients with Alport syndrome
who develop post-transplant nephritis. Previous work has established
that Goodpasture autoantibodies are also targeted to the NC1 domain of
the 3(IV) chain (1). The Alport alloantibodies, produced in some
patients in response to a renal transplant, are targeted to the NC1
domain of the
3(IV) chain in certain patients (59, 60) and the
5(IV) chain in others (21, 61, 62). As shown in Fig. 8D,
both GP autoantibodies (63) and Alport alloantibodies (59) bind to
fraction I of the 25 °C protomers. Thus, the
3(IV)·
4(IV)·
5(IV) network shown in Fig. 6A is
the target for both kinds of antibodies that cause anti-GBM
nephritis.
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ACKNOWLEDGEMENTS |
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We thank Parvin Todd and Billy J. Wisdom, Jr. for skillful assistance, and Dr. Raghu Kalluri for assistance in electrophoresis and blotting.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK 18381. A preliminary report of this work has been presented elsewhere (Gunwar, S., Ballester, F., Noelken, M. E., and Hudson, B. G. (1992) J. Am. Soc. Nephrol. 3, 296).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
Biochemistry and Molecular Biology, University of Kansas Medical
Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421.
1 The abbreviations used are: LBM, lens basement membrane; GBM, glomerular basement membrane; STBM, seminiferous tubule basement membrane; NC1, noncollagenous domain; HPLC, high pressure liquid chromatography; GP, Goodpasture; ALP, Alport; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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