Glomerular Basement Membrane
IDENTIFICATION OF A NOVEL DISULFIDE-CROSS-LINKED NETWORK OF alpha 3, alpha 4, AND alpha 5 CHAINS OF TYPE IV COLLAGEN AND ITS IMPLICATIONS FOR THE PATHOGENESIS OF ALPORT SYNDROME*

Sripad GunwarDagger , Fernando BallesterDagger , Milton E. NoelkenDagger , Yoshikazu Sado§, Yoshifumi Ninomiya, and Billy G. HudsonDagger par

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 1(IV) and alpha 2(IV) chains, whereas the 25 °C protomers comprised mainly alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains along with some alpha 1(IV) and alpha 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 alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 5(IV) chain cause defective assembly of not only alpha 5(IV) chain but also the alpha 3(IV) and alpha 4(IV) chains in the GBM of patients with Alport syndrome.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha (IV) chains (1). These are designated alpha 1(IV) to alpha 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 alpha 1(IV) and alpha 2(IV) chains have a ubiquitous distribution and the alpha 3(IV) to alpha 6(IV) chains have a restricted distribution (2-5). For example, LBM is composed mainly of the alpha 1(IV) and alpha 2(IV) chains (6) and STBM is composed of all six alpha (IV) chains (7), whereas GBM is composed of five chains, alpha 1(IV) to alpha 5(IV) (5, 6, 8). The chains are assembled into triple-helical molecules (protomers, Fig. 1) composed of three alpha (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 [alpha 1(IV)]2 alpha 2(IV) (9) and an [alpha 3(IV)]2 alpha 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 alpha 3(IV) and alpha 4(IV) chains (11). Three such networks have been identified: a ubiquitous alpha 1(IV)·alpha 2(IV) network and both an alpha 1(IV)-alpha 6(IV) and an alpha 3(IV)-alpha 6(IV) network in STBM (7). An alpha 3(IV)-containing network, that does not contain alpha 1(IV) and alpha 2(IV) chains, has been identified in GBM (10, 12).

In Alport syndrome, mutations occur in the COL4A3, COL4A4, and COL4A5 genes encoding the alpha 3(IV), alpha 4(IV), and alpha 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 alpha 3(IV) and alpha 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 alpha 1(IV) and alpha 2(IV) chains and is devoid of the alpha 3(IV), alpha 4(IV), and alpha 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 alpha 1(IV) and alpha 2(IV) chains persist and are not replaced by the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains that are necessary to form a mature GBM (22). How mutations in the alpha 5(IV) chain cause defective assembly of not only the alpha 5(IV) chain, but also the alpha 3(IV) and alpha 4(IV) chains, remains a conundrum. The mechanism linking the assembly of these three chains could be at the protein level in which the alpha 3(IV), alpha 4(IV), and alpha 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 alpha 3(IV), alpha 4(IV), and alpha 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 alpha 3(IV), alpha 4(IV), and alpha 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 alpha 3(IV), alpha 4(IV), and alpha 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 alpha 5(IV) chain, but also the alpha 3(IV) and alpha 4(IV) chains.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 1(IV) and alpha 2(IV) chains and the pellet contained primarily alpha 3(IV), alpha 4(IV), and alpha 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 alpha -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 alpha (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 alpha 1(IV)-alpha 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 alpha (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).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (alpha 1 to alpha 6) of alpha (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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Fig. 1.   Structure of type IV collagen. The protomer contains three alpha (IV) chains and is triple-helical over most of its length. There are six genetically distinct alpha (IV) chains. The number of protomers that can be made in vivo is not known. Each chain contains a carboxyl-terminal noncollagenous NC1 and an amino-terminal 7 S domain; the latter contains a large Asn-linked oligosaccharide (Y-shaped structure). Protomers associate through NC1 domains and 7 S domains to form suprastructures. Digestion of basement membranes with pseudolysin yields truncated protomers that contain the NC1 domains and part of the triple helix.

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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Fig. 2.   Gel filtration chromatography (Sephacryl S-1000) of truncated protomers of type IV collagen that were obtained from sequential digestion of GBM at 4 °C and 25 °C with pseudolysin. Elution profiles of solubilized GBM; the digestion temperature is indicated for each profile. In each case, fractions 20-120 were pooled, concentrated by ultrafiltration, and used for subsequent experiments. The arrow designates where pseudolysin begins to elute. Inset, electrophoretic pattern (SDS-PAGE) of the pooled fractions from the 4 °C and 25 °C digests under nonreducing conditions, and the molecular weight markers with the size in kilodaltons (kDa).

Chain Composition of 4 °C and 25 °C Protomers-- The identity of alpha (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-alpha 1(IV) and alpha 2(IV) antibodies, with no reactivity to antibodies directed against alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains (Fig. 3A). These results indicated that pseudolysin cleaved and selectively solubilized the alpha 1(IV) and alpha 2 chains of GBM at 4 °C. The NC1 domains of the 25 °C protomers showed reactivity to antibodies directed against alpha 1(IV), alpha 2(IV), alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, and no reactivity toward the alpha 6(IV) chain (Fig. 3B). This indicated that the 25 °C protomers were mainly composed of the alpha 3(IV), alpha 4(IV), and alpha 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 alpha 3(IV) NC1 domain (1, 7).


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Fig. 3.   Identification of the NC1 domains comprising the 4 °C and 25 °C truncated protomers. NC1 domains were prepared from the 4 °C and 25 °C protomer fractions (20-120, fraction volume = 5.2 ml) from the Sephacryl S-1000 column shown in Fig. 2 by collagenase digestion at 37 °C. The NC1 domains were analyzed by SDS-PAGE under non-reducing conditions as described previously (38), with Western blotting with chain-specific monoclonal antibodies (panel A, 4 °C protomers; panel B, 25 °C protomers). CB indicates protein staining with Coomassie Blue. The alpha (IV) chain specificity of antibodies is noted on the lanes (alpha 1-alpha 6). GP indicates blotting with Goodpasture autoantibodies, and ALP indicates blotting with Alport alloantibodies. D and M represent monomers and dimers (63).

The NC1 domains of the 4 °C and 25 °C protomers were also analyzed by HPLC and two-dimensional electrophoresis to identify and quantitate the kinds of chains (Fig. 4). The HPLC profile and two-dimensional electrophoresis pattern of whole GBM (Fig. 4, A and D, respectively) were similar to previous results (7). The composition of the 4 °C and 25 °C protomers, based on HPLC analysis, are presented in Table I. The ratios are consistent with the two-dimensional electrophoresis patterns which show that the NC1 domains of the 4 °C protomers are mainly derived (>90%) from the alpha 1(IV) and alpha 2(IV) chains, and the NC1 domains (monomer and dimer) of the 25 °C protomers are mainly derived from the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, with minor amounts from the alpha 1(IV) and alpha 2(IV) chains. Further experiments (data not shown) demonstrated that pseudolysin digestion at 25 °C did not completely remove alpha 1(IV) and alpha 2(IV) chains from GBM, even when the digestion was extended to >50 h and with the addition of fresh pseudolysin. Overall, these results indicate that the 4 °C protomers mainly comprise (>90%) the alpha 1(IV) and alpha 2(IV) chains, whereas the 25 °C protomers mainly comprise (>90%) the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, along with a small amount (<10%) of alpha 1(IV) and alpha 2(IV) chains.


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Fig. 4.   Quantitation of the alpha (IV) NC1 domains comprising the 4 °C protomers and 25 °C protomers. The NC1 domains were prepared by collagenase digestion of samples and were analyzed by HPLC (panels A-C) and two-dimensional electrophoresis (panels D-F). The first dimension was run under non-reducing conditions in the presence of 6 M urea and the second dimension was run under non-reducing conditions in the presence of 1% SDS as described previously (27). Panels A and D correspond to whole GBM, B and E correspond to 4 °C protomers, and C and F to 25 °C protomers. The NC1 domains were resolved into fractions I to IV by HPLC (panels A-C). The composition of fractions is as follows: fraction I = alpha 1(IV), alpha 2(IV), and alpha 5(IV) monomers; fraction II = alpha 1(IV) and alpha 2(IV) dimers; fraction III = alpha 4(IV) monomers and dimers; and fraction IV = alpha 3(IV) monomers and dimers (7). The arrowheads designate pI markers (panels D-F), and the location of the various NC1 domains was established previously (7). Panel E shows that the NC1 domain of the 4 °C protein corresponds to monomers and dimers of alpha 1(IV) and alpha 2(IV) NC1 domains, according to the migration positions identified previously (63). In contrast, panel F shows that NC1 domains of the 25 °C protomers correspond to monomers and dimers of the alpha 3(IV), alpha 4(IV), and alpha 5(IV) NC1 domains.

                              
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Table I
Chain composition of 4 °C and 25 °C protomers

Supramolecular Organization of 4 °C and 25 °C Protomers

Rotary Shadowing Electron Microscopy-- The 4 °C truncated protomers, comprising the alpha 1 and alpha 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 alpha 1(IV)·alpha 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 alpha 1(IV)·alpha 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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Fig. 5.   Electron microscopic analysis of 4 °C protomers. Panel A shows the major component, a dimer of truncated protomers that have two triple helices of 300 ± 28 nm contour length originating from a central NC1 domain hexamer 16 nm in diameter. The bar represents 100 nm. Panel B, a low percentage of the fragments have triple helices of contour length only 140 nm; these occurred primarily as dimers but monomers (data not shown) are also present. Panel C, a low percentage of fragments are dimers with one triple helix 300 nm long and the other one 140 nm long. Panel D, examples of fragments that apparently originated from the 7 S domain.

The 25 °C protomers, mainly comprising the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, were also analyzed by electron microscopy. These exhibited a complex supramolecular structure characterized by looping and supercoiling of the triple-helical domains (Fig. 6A). Pepsin digestion removed the NC1 domains from the 25 °C protomers but did not alter the supercoiling and loop structures of the triple-helical domain (Fig. 6B). Reduction and alkylation under non-denaturing conditions effected substantial conversion of the suprastructures into truncated protomers (monomers and NC1-NC1 linked dimers) (Fig. 7A). Reduction and alkylation under non-denaturing conditions of a pepsin digested sample resulted in the conversion of most of the complexes into relatively simple molecules that lacked NC1 domains (Fig. 7B). Overall, these results indicate that the suprastructures of the 25 °C protomers are stabilized by disulfide cross-links between triple-helical domains and between NC1 domains of adjoining protomers.


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Fig. 6.   Electron microscopic analysis of 25 °C protomers. Panel A, the 25 °C protomers were from Sephacryl S-1000 column (Fig. 2); bar represents 100 nm. Panel B, the protomers were digested with pepsin; bar represents 100 nm.


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Fig. 7.   Electron microscopic analysis of 25 °C protomers after reduction of disulfide bonds. Panel A, the protomers shown in Fig. 6A were reduced and alkylated under non-denaturing conditions; bar represents 100 nm. Panel B, the pepsin-digested protomers shown in Fig. 6B were reduced and alkylated under non-denaturing conditions; bar represents 100 nm.

Identification of a Disulfide-linked Complex of the alpha 3(IV), alpha 4(IV), and alpha 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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Fig. 8.   Characterization chain constituents of the 25 °C protomers. Panel A, the protomers were analyzed by SDS-agarose electrophoresis to determine the size of components dissociated in SDS with Mr >200,000. Lane a, calf skin collagen I; lane b, A. suum intestinal basement membrane type IV collagen; lane c, rabbit skeletal muscle myosin were used as size markers. The numbers 200, 515, and 1080 are the sizes in kDa for myosin and the two fragments of A. suum intestinal basement membrane type IV collagen, respectively. Lane e corresponds to the 25 °C protomers shown in Fig. 5A, lane f corresponds to the reduced and alkylated protomers shown in Fig. 7A, and lane g corresponds to the pepsin-digested protomers that were reduced and alkylated (Fig. 7B). Lane d corresponds to fraction I of Fig. 8B. In panel B, the 25 °C protomers shown in Fig. 6A were dissociated in 6 M guanidine-HCl and fractionated on a Sephacryl S-400 column. Fraction I was pooled for further study. In panel C, fraction I was analyzed by SDS-PAGE before (NR) and after reduction (R) with 2-mercaptoethanol. Upon reduction the constituents penetrate the resolving gel. The constituents were identified by Western blotting with alpha (IV) chain-specific antibodies and their length in amino acid residues is shown. The Mr of standard proteins is given in kDa. In panel D, the NC1 domains of fraction I were released by collagenase digestion and their alpha (IV) chain identities were determined by SDS-PAGE with Western blotting using alpha (IV) chain-specific antibodies. Fraction I reacted with alpha 3(IV), alpha 4(IV), and alpha 5(IV) monoclonal antibodies and GP (patient LL) and ALP antibodies. CB indicates protein staining with Coomassie Blue. The mobility of molecular weight markers is shown.

The 25 °C protomers were also denatured in 6 M guanidine-HCl and fractionated on a column of Sephacryl S-400 (Fig. 8B) to identify disulfide cross-linked components. Fraction I, the major fraction, corresponds to the high molecular weight component(s) (Mr 500,000) observed by SDS-agarose electrophoresis (Fig. 8A, lane d (fraction I versus lane e (unfractionated)). When analyzed by SDS-PAGE, fraction I did not penetrate the stacking gel because of its large size, but upon complete reduction of disulfide bonds, it dissociated into multiple polypeptides with apparent Mr <160,000 (Fig. 8C). Fractions II and III (Fig. 8B) consisted of components of lower molecular weight than fraction I, and both yielded components (Mr <160,000) upon reduction (data not shown). Overall, these results indicate that fraction I is a high molecular weight component(s), composed of truncated alpha (IV) chains that are cross-linked by disulfide bonds between triple-helical domains.

The identity of alpha (IV) chains comprising fractions I, II, and III was determined from the identity of the NC1 domains that were released upon collagenase digestion. The NC1 domains were identified by SDS-PAGE with Western blotting, using chain-specific antibodies. Fraction I reacted with anti-alpha 3(IV), -alpha 4(IV), and -alpha 5(IV) antibodies and not with anti-alpha 1(IV), -alpha 2(IV), or -alpha 6(IV) antibodies (Fig. 8D). In contrast, fractions II and III reacted with alpha 1(IV) to alpha 5(IV) antibodies but not with alpha 6(IV) antibodies (data not shown). The alpha 4(IV) and alpha 5(IV) antibodies reacted primarily with dimers in fraction I, but with monomers in fractions II and III. Six molar guanidine-HCl is a denaturing and dissociating agent, and its ability to dissociate the complexes shows that there are noncovalent interactions between alpha 1(IV) and alpha 2(IV) chain fragments and the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chain fragments. The failure of 6 M guanidine-HCl to dissociate fraction I into its constituent alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, which were identified by SDS-PAGE after reduction (Fig. 8C), indicated that they exist as a high molecular weight complex in which they are cross-linked by disulfide bonds. Moreover, this alpha 3(IV)·alpha 4(IV)·alpha 5(IV) complex serves as the target antigen for both GP autoantibodies and ALP alloantibodies (Fig. 8D).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 1(IV) and alpha 2(IV) chains. By electron microscopy, they exhibited structural characteristics of alpha 1(IV)·alpha 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 alpha 1(IV) and alpha 2(IV) chains in GBM. The existence of such a network is also supported by the finding that NC1 hexamers comprising alpha 1(IV) and alpha 2(IV) NC1 domains can be isolated from GBM (10, 12), although the cited studies could not rule out the presence of alpha 5(IV) and alpha 6(IV) chains.

In contrast to the truncated protomers solubilized at 4 °C, those solubilized at 25 °C were mainly comprising the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, along with a small amount of alpha 1(IV) and alpha 2(IV) chains. These exhibited some structural features that are in common with the alpha 1(IV)·alpha 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 alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains of the complex are covalently linked by disulfide bonds, exclusive of alpha 1(IV) and alpha 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 alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. Fragments of the alpha 1(IV) and alpha 2(IV) chains could be separated from the alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV), alpha 4(IV), and alpha 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 alpha 3(IV) and alpha 4(IV) NC1 domains, can be isolated from GBM (10), although the studies did not address the presence of the alpha 5(IV) and alpha 6(IV) chains. Moreover, an alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network is consistent with immunocytochemical results showing that the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains, but not the alpha 6(IV) chain, are present in GBM (8, 33). A similar network that also contains the alpha 6(IV) chain is present in STBM (7).

A third network comprising alpha 1(IV)-alpha 5(IV) chains may also exist. The 25 °C protomers contain some alpha 1(IV) and alpha 2(IV) fragments that could be noncovalently associated with the alpha 3(IV), alpha 4(IV), and alpha 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 alpha 1(IV) and alpha 2(IV) chains as well as alpha 3(IV) and alpha 4(IV) chains (10). Moreover, the residual insoluble GBM from 25 °C pseudolysin digestion contains alpha 1(IV)-alpha 5(IV) chains, which could comprise an alpha 1(IV)-alpha 5(IV) network. Such a network that also contains the alpha 6(IV) chain exists in STBM (7). All three networks alpha 1(IV)·alpha 2(IV), alpha 3(IV)·alpha 4(IV)·alpha 5(IV), and alpha 1(IV)-alpha 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 alpha 3(IV)·alpha 4(IV)·alpha 5(IV) truncated protomers is a distinguishing feature not found in the alpha 1(IV)·alpha 2(IV) truncated protomers. This can be rationalized by the presence of several cysteine residues in the collagenous domain of the alpha 3(IV) and alpha 4(IV) chains that are not found in the alpha 1(IV) and alpha 2(IV) chains. Comparison of the amino acid sequences of the six alpha (IV) chains (39-48) reveals the following. First, there are 18 conserved cysteine residues among all six human alpha (IV) chains: 4 in the 7 S domain, 12 in the NC1 domain, and 2 in triple-helical interruption IX (Fig. 9). Second, the alpha 3(IV) chain contains four cysteine residues and the alpha 4(IV) chain contains nine cysteine residues within the collagenous domain that are not found in the other four alpha (IV) chains (Fig. 9). Third, the truncated alpha 3(IV) and alpha 4(IV) chains that are constituents of the alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV)·alpha 4(IV)·alpha 5(IV) complex shown in Fig. 7A. Fourth, the truncated alpha 5(IV)chain of the alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV) or alpha 4(IV) chain, either within a protomer or between protomers. The alpha 5(IV) chain is also linked to the alpha 3(IV) or alpha 4(IV) chain through disulfide bonds between NC1 domains because the 1100-residue fragment of the alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network.


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Fig. 9.   Schematic diagram of collagen IV chains. In each chain, the hatched region indicates a putative signal peptide; the filled regions are noncollagenous domains, with the larger one representing the NC1 domain, and the intervening region is the major collagenous domain. The interruptions of the Gly-Xaa-Yaa repeating sequence of the major collagenous domain are indicated by vertical lines. The approximate location of cysteine residues is indicated by the letter C, with C in interruptions placed above the chain and those in triple helices placed below the chain. The alpha 1(IV), alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains have 1669, 1712, 1670, 1690, 1685, and 1691 residues, respectively. The approximate positions of some pseudolysin cleavage points are indicated by arrows, and these are based on the size of the truncated chains shown in Fig. 8C.

The alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 1(IV) and alpha 2(IV) chains but which were studied before it was possible to detect alpha 3(IV), alpha 4(IV), and alpha 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 alpha 1(IV) and alpha 2(IV) chains, could be induced to form a polygonal network (49). The alpha 3(IV)·alpha 4(IV)·alpha 5(IV) supramolecular structure that we observed is stabilized by disulfide cross-links between the constituent alpha (IV) chains. Conceivably, the alpha 1(IV)·alpha 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 alpha 1(IV)·alpha 2(IV) network and an alpha 3(IV)· alpha 4(IV)·alpha 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 alpha 1(IV) and alpha 2(IV) chains is replaced by the expression of the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains to form a mature GBM (20, 54). In X-linked Alport syndrome, the switch is arrested, causing the persistence of the alpha 1(IV) and alpha 2(IV) chains and the absence of the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains (20). Based upon the present results, the switch can now be defined at the supramolecular level in which the alpha 1(IV)·alpha 2(IV) network is replaced by the alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network in normal glomerular development, but in Alport syndrome the switch is arrested. Second, it is well established that mutations in the alpha 5(IV) chain cause defective assembly of not only the alpha 5(IV) chain, but the alpha 3(IV) and alpha 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 alpha (IV) chains. The finding of an alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network, in which all three chains are covalently linked by disulfide cross-links, provides a possible linkage mechanism, wherein the alpha 5(IV) chain is required for the assembly of the alpha 3(IV) and alpha 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 alpha 1(I) chain cause defective assembly of the alpha 2(I) chain (55). The alpha 5(IV) chain requirement could be at: (a) the protomer level in which an alpha 5(IV) chain together with an alpha 3(IV) and alpha 4(IV) chain forms a triple-helical protomer or (b) the supramolecular level in which an alpha 5(IV)-containing protomer is required for the incorporation of an alpha 3(IV) or alpha 4(IV) containing protomer. The linkage mechanism may also involve the transcriptional/translational level because the mRNA expression of the alpha 3(IV), alpha 4(IV), and alpha 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 alpha 5(IV) chain into extracellular matrix could be required to modulate the transcription of the alpha 3(IV) and alpha 4(IV) chains.

The alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are absent in the GBM (56, 57). Thus, the alpha 3(IV) chain is crucial for the assembly of the alpha 4(IV) and alpha 5(IV) into an alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network.

The fundamental importance of the alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network for normal glomerular ultrafiltration function is evident from the gene mutations that cause renal failure in Alport syndrome and from the alpha 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 alpha 1(IV)·alpha 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 alpha 1(IV)·alpha 2(IV) network is more easily excised from GBM by pseudolysin than the alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network as described herein, which further supports the hypothesis that the alpha 3(IV)·alpha 4(IV)·alpha 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 alpha 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 alpha 3(IV) chain in certain patients (59, 60) and the alpha 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 alpha 3(IV)·alpha 4(IV)·alpha 5(IV) network shown in Fig. 6A is the target for both kinds of antibodies that cause anti-GBM nephritis.

    ACKNOWLEDGEMENTS

We thank Parvin Todd and Billy J. Wisdom, Jr. for skillful assistance, and Dr. Raghu Kalluri for assistance in electrophoresis and blotting.

    FOOTNOTES

* 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.

par 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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Abstract
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Results
Discussion
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