NC1 Hexamer Research Articles

Type IV Collagen of the Glomerular Basement Membrane: EVIDENCE THAT THE CHAIN SPECIFICITY OF NETWORK ASSEMBLY IS ENCODED BY THE NONCOLLAGENOUS NC1 DOMAINS

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (alpha1 to alpha5) of the six chains of type IV collagen, organized into an alpha1.alpha2(IV) and an alpha3.alpha4.alpha5(IV) network. In Alport syndrome, mutations in any of the genes encoding the alpha3(IV), alpha4(IV), and alpha5(IV) chains cause the absence of the alpha3. alpha4.alpha5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the alpha5 chain was shown to be linked to the alpha3 and alpha4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified alpha1-alpha5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an alpha1.alpha2 and alpha3.alpha4.alpha5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the alpha1.alpha2 and alpha3.alpha4.alpha5 networks of GBM. Moreover, hexamer formation from the alpha3, alpha4, and alpha5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the alpha3.alpha4.alpha5 network by interfering with the assembly of the alpha3.alpha4.alpha5 NC1 hexamer.

Reactive Oxygen Species Expose Cryptic Epitopes Associated with Autoimmune Goodpasture Syndrome

Goodpasture syndrome is an autoimmune disease of the kidneys and lungs mediated by antibodies and T-cells directed to cryptic epitopes hidden within basement membrane hexamers rich in alpha3 non-collagenous globular (NC1) domains of type IV collagen. These epitopes are normally invisible to the immune system, but this privilege can be obviated by chemical modification. Endogenous drivers of immune activation consequent to the loss of privilege have long been suspected. We have examined the ability of reactive oxygen species (ROS) to expose Goodpasture epitopes buried within NC1 hexamers obtained from renal glomeruli abundant in alpha3(IV) NC1 domains. For some hexameric epitopes, like the Goodpasture epitopes, exposure to ROS specifically enhanced recognition by Goodpasture antibodies in a sequential and time-dependent fashion; control binding of epitopes to alpha3(IV) alloantibodies from renal transplant recipients with Alport syndrome was decreased, whereas epitope binding to heterologous antibodies recognizing all alpha3 NC1 epitopes remained the same. Inhibitors of hydrogen peroxide and hydroxyl radical scavengers were capable of attenuating the effects of ROS in cells and kidney by 30-50%, respectively, thereby keeping the Goodpasture epitopes largely concealed when compared with a 70% maximum inhibition by iron chelators. Hydrogen peroxide administration to rodents was sufficient to expose Goodpasture epitope in vivo and initiate autoantibody production. Our findings collectively suggest that ROS can alter the hexameric structure of type IV collagen to expose or destroy selectively immunologic epitopes embedded in basement membrane. The reasons for autoimmunity in Goodpasture syndrome may lie in an age-dependent deterioration in inhibitor function modulating oxidative damage to structural molecules. ROS therefore may play an important role in shaping post-translational epitope diversity or neoantigen formation in organ tissues.

Chain composition of type IV collagen networks in basement membranes

Type IV collagen, the major component of basement membranes (BM), is a family of six very similar chains. This allows for many kinds (isoforms) of triple helical protomers. The protomers self-assemble into network-like supramolecular structures by dimerization through C-terminal (NCl) domains (resulting in six chains forming an NC1 hexamer), and tetramerization through the protomer N-terminal domain. Studies on the networks of seminiferous tubule BM (STBM) involved affinity chromatography to fractionate NCl hexamers excised by collagenase digestion. Three major hexamer populations were obtained that containedα1(IV) andα2(IV) chains,α1(IV)-α6(IV) chains, andα3(IV)-α6(IV) chains, respectively, which indicated three major separate type IV collagen networks in STBM. To study glomerular BM, we used selective proteolysis to separate large fragments of type IV collagen. These contained type IV collagen chains cross-linked between NCl domains, andα1(IV) throughα5(IV) chains cross-linked between NC1 domains as well between triple helices. The latter set of fragments contained a subset of disulfidelinkedα3(IV),α4(IV) andα5(IV) chains. The existence of networks containingα3(IV),α4(IV) andα5(IV) chains suggests a molecular basis for mutations in the gene encoding theα5(IV) chain causing defective assembly of not onlyα5(IV) chains, but alsoα3(IV) andα4(IV) chains in the GBM of patients with Alport syndrome. A significant result of these studies is that GBM contains three major type IV collagen networks:α1(IV) ·α2(IV),α1(IV)-α5(IV) andα3(IV) ·α4(IV) ·α5(IV), similar to those of STBM, which also containsα6(IV) chains in the latter two networks.

Experimental Goodpasture's syndrome in Wistar-Kyoto rats immunized with α3 chain of type IV collagen

Glomerulonephritis and lung hemorrhage of autoimmune Goodpasture syndrome develop due to immune reactions against epitope(s) of the non-collagenous (NC1) domain of alpha3-chain of type IV collagen [alpha3(IV) NC1]. Whether thymic mechanisms have a role in the loss of tolerance to the Goodpasture epitope has not been established. We studied the renal and pulmonary effects of immunization with different forms (monomer, dimer, or hexamer) of alpha3(IV) NC1 collagen in Wistar-Kyoto (WKY) rats, and assessed whether the intrathymic inoculation of the antigen may protect against anti-GBM disease. WKY rats were immunized with bovine alpha3(IV) monomer, dimer, or hexamer, or with alpha3(IV) NC1 synthetic peptide. Renal function, kidney and lung immunohistology, and circulating and tissue bound antibodies to type IV collagen chains were analyzed. Effects of intrathymic inoculation of antigen on subsequent disease induction were analyzed in WKY rats given alpha3(IV) NC1 dimer or GBM preparation intrathymically 48 hours before immunization. Proteinuria, linear IgG deposition in GBM, and crescentic glomerulonephritis developed in WKY rats immunized with alpha3(IV) NC1 dimer or hexamer. Lesions were dose-dependent upon injections of 10 to 100 microgram dimer. The alpha3(IV) NC1 monomer induced less severe proteinuria and no crescents. Pulmonary hemorrhage was detectable in 35% of rats immunized with 25 to 100 microgram alpha3(IV) NC1 dimer; alpha3(IV) synthetic peptide (36 carboxyl terminal) did not induce disease. Rats injected intrathymically with up to 100 microgram alpha3(IV) NC1 dimer or with GBM 48 hours before immunization were not protected against subsequent development of proteinuria and glomerulonephritis. These findings document that glomerulonephritis and lung hemorrhage can be elicited in WKY rats by immunization with alpha3(IV) NC1. Failure of the intrathymic inoculation of antigen to prevent disease suggests that immunological tolerance cannot be achieved by this intervention, in contrast to other autoimmune conditions, and may imply independent roles for cellular and humoral nephritogenic pathways in anti-GBM nephritis.

Seminiferous tubule basement membrane. Composition and organization of type IV collagen chains, and the linkage of alpha3(IV) and alpha5(IV) chains.

Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six alpha-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of alpha3(IV) chain (40%) and the alpha4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six alpha(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of alpha(IV) chains, were characterized for their alpha(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with alpha(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the alpha1(IV) and alpha2(IV) chains and two novel networks, one composed of the alpha1(IV)-alpha6(IV) chains and the other composed of the alpha3(IV)-alpha6(IV) chains. The results establish a structural linkage between the alpha3(IV) and alpha5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of the alpha3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.

The Goodpasture Autoantigen: STRUCTURAL DELINEATION OF TWO IMMUNOLOGICALLY PRIVILEGED EPITOPES ON α3(IV) CHAIN OF TYPE IV COLLAGEN

The family of type IV collagen comprises six chains numbered alpha1 through alpha6. The alpha3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the alpha3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human alpha3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the alpha3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated alpha3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant alpha3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the alpha3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the alpha3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the alpha3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the alpha3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.

The alpha 3 chain of type IV collagen induces autoimmune Goodpasture syndrome.

Human Goodpasture syndrome is a lethal form of autoimmune disease that is characterized by pulmonary hemorrhage and glomerulonephritis. The tissue injury is mediated by autoantibodies that bind to glomerular and alveolar basement membrane. The target autoantigen is alpha 3(IV) collagen, one of six genetically distinct chains that comprise type IV collagen, and the epitope is sublocalized to the noncollagenous domain (NC1) of the alpha 3 chain. The present study reports the unique capacity of alpha 3(IV)NC1 dimer from bovine kidney to aberrantly engage the immune system of rabbits to respond to self, mimicking the organ-specific form of the human disease, whereas the other chains of type IV collagen are nonpathogenic. However, alpha 3(IV)NC1 hexamer was nonpathogenic, suggesting the exposure of a pathogenic epitope upon dissociation of hexamer into dimers. Exposure of the pathogenic epitope by infection or organic solvents, events which are thought to precede Goodpasture syndrome, may be the principal factor in the etiology of the disease. The pathogenicity of alpha 3(IV) collagen brings full circle a decade of research that has identified four novel chains (alpha 3-alpha 6) of type IV collagen.

The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane.

Type IV collagen, which has long been assumed to contain two alpha 1(IV) and one alpha 2(IV) chains, also contains alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. Stoichiometry of collagenous alpha(IV) chains differs among tissues, suggesting the existence of subclasses of type IV collagen, each with a unique chain composition. This study seeks to define, by characterization of subunit compositions of NC1 domain populations, the structural organization of type IV collagen from bovine glomerular basement membrane. NC1 hexamers from type IV collagen were separated on two affinity chromatography columns, one containing monoclonal antibodies to the alpha 3 chain, and another, to the alpha 1 chain. SDS-polyacrylamide gel electrophoresis, immunoblotting, reversed phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay identified three NC1 hexamer populations: 1) a hexamer composed of (alpha 1)2 and (alpha 2)2 homodimers; 2) a hexamer composed of (alpha 3)2 and (alpha 4)2 homodimers; 3) a hexamer containing all four alpha chains connected in heterodimers, alpha 1-alpha 3 and alpha 2-alpha 4. Results suggest that there are two distinct type IV collagen molecules, one composed of alpha 1(IV) and alpha 2(IV) chains and another composed of alpha 3(IV) and alpha 4(IV) chains. Furthermore, polymerization occurs between molecules with the same chain composition and between molecules with different chain composition. Moreover, crosslinking between different alpha chains is restricted, thus limiting the number of possible macromolecular structures.

Different immunologic properties of the globular NC1 domain of collagen type IV isolated from various human basement membranes.

The C-terminal globular domain NC1 of collagen IV, which carries the epitopes recognized by anti-GBM antibodies in Goodpasture's syndrome, was isolated from human basement membranes (BM) of glomeruli (GBM-NC1), tubules (TBM-NC1), lung (ABM-NC1), placenta (PBM-NC1), and small intestine (IBM-NC1). All NC1 hexamers were of globular size on electron microscopy. On SDS-PAGE, the hexamers dissociated into monomeric and dimer-sized subunits of similar molecular weights. The following monomer:dimer relationships were identified: GBM-NC1, and PBM-NC1 = 1:3; ABM-NC1 = 1:4; and IBM-NC1 = 1:32. On immunoblot, all dimers of the various NC1 globules showed binding of anti-GBM antibodies. However, monomers stained differently, with three monomers demonstrable in GBM-NC1 and no monomer staining in PBM-NC1. In addition, studies with monoclonal antibodies showed that the C-terminus of the alpha 1(IV) collagen chain was demonstrable in all different NC1 hexamers. In contrast, the alpha 3(IV) chain, to which Goodpasture sera preferentially bind, showed a restricted distribution. One monomer and dimers were demonstrable in GBM-NC1 and ABM-NC1, only a weak dimer staining was seen in TBM-NC1, while no evidence for alpha 3(IV) was found in IBM-NC1 and PBM-NC1. Dissociation by 6 M guanidine-HC1 or treatment by acid increases the apparent number of accessible epitopes for anti-GBM antibodies. In addition, dose-response curves, which were obtained by incubation of increasing concentrations of NC1 with anti-GBM antibody positive sera, indicated that for GBM-NC1 and ABM-NC1 the lowest NC1 protein concentrations were necessary to bind 50% of the antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Type IV collagen of Engelbreth-Holm-Swarm tumor matrix: identification of constituent chains.

The noncollagenous hexamer (NC1) domain of type IV collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix was subjected to electrophoretic, amino-terminal amino acid sequence, and immunochemical analysis to determine which of the five known kinds of alpha(IV) chains are present. Electrophoretic analysis, whether by one-dimensional or two-dimensional electrophoresis, showed that nonlathyritic and lathyritic hexamer gave nearly identical patterns. Amino-terminal amino acid sequence analysis of hexamer subunits, transblotted from two-dimensional gels, revealed that the hexamer subunits were derived exclusively from the alpha 1 and alpha 2 chains. Western blots of hexamer subunits confirmed the sequence results, as the subunits. identified as alpha 1(IV) and alpha 2(IV) NC1 domains reacted with antibodies directed specifically against those subunits. Conversely, no reactivity of NC1 hexamer subunits was seen with Goodpasture serum, or with antibodies directed specifically against the alpha 3, alpha 4, and alpha 5 NC1 domains, confirming the lack of alpha 3, alpha 4, and alpha 5 chains. These results revealed that the type IV collagen component of the EHS sarcoma matrix is comprised exclusively of alpha 1 and alpha 2 chains. Its relative homogeneity simplifies, but restricts, interpretation of studies that employ it as a model type IV collagen because the studies would be based only on alpha 1 and alpha 2 chains.

Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV.

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.

Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV).

The formation of collagen IV dimers in the extracellular space requires the association of two C-terminal globular domains giving rise to a large hexameric structure NC1 (Mr = 170,000). NC1 hexamer was purified from collagenase digests of a mouse tumor and several human tissues. It was shown by electrophoresis to consist of two kinds of cross-linked, dimeric segments, Da and Db (Mr about 50,000), and monomeric segments in a molar ratio of about 3:1. In the native hexamers free SH groups were detectable by N-[14C]ethylmaleimide and other sulfhydryl reagents. They account for 4-11% of the total number of cysteine residues with some variations between preparations from different sources and in the distribution between monomers and dimers. Reduction with 10 mM dithioerythritol under non-denaturing condition completely converted dimers into monomers and allowed the alkylation of all twelve cysteine residues present in each monomeric NC1 segment. A monomeric intermediate with four to six free SH groups and a higher electrophoretic mobility than the final product was observed. Generation of this intermediate from dimers Da and Db follows apparently different routes proceeding either directly or through a dimeric intermediate respectively. The time course of conversion is best described by a mechanism consisting of two (Db) or three (Da) consecutive steps with pseudo-first-order rate constants ranging from 0.14 ms-1 to 0.5 ms-1. Glutathione-catalyzed reoxidation of completely reduced NC1 in the presence of 2 M urea results in a product indistinguishable from native material by ultracentrifugation and electrophoresis pattern. The data suggest that in situ formation of NC1 structures is catalyzed by a small fraction (5-10%) of intrinsic SH groups leading to the formation and stabilization of dimers by rearrangement of disulfide bonds.

Comparison of non-collagenous type IV collagen components in the human glomerulus and EHS tumor

A method for the isolation of the NC1 domain of type IV collagen has been developed using the EHS sarcoma, a basement membrane-producing mouse tumor. This NC1 domain has been compared to the NC1 of human glomerular basement membrane (hGBM) to assess its usefulness in the biochemical characterization of the Goodpasture antigen which is associated with NC1. Both NC1 isolates appeared to migrate by gel filtration as hexamers ( M r 160 000) and in SDS-polyacrylamide gel electrophoresis as dimers and monomers ( M r 54 000 and 26 000), and were shown to share biochemical identity by amino acid analysis. The hGBM NC1 showed greater complexity in the monomer region, and when compared by two-dimensional gel electrophoresis was found to contain more components in both regions than EHS NC1. Anti-GBM autoantibodies from patients with Goodpasture's syndrome reacted with the EHS NC1 by immunoblotting of two-dimensional gels. The EHS NC1 isolated by reverse phase HPLC partially inhibited the reactivity of the anti-GBM autoantibodies against hGBM NC1 by inhibition ELISA assay. Reverse phase HPLC elution of EHS and hGBM NC1 showed differences in subunit composition and interaction; complete dissociation of the EHS monomers and dimers in 0.1% trifluoroacetic acid was observed, whereas hGBM monomers and dimers eluted together. Rotary shadowing of hGBM NC1 domains revealed size heterogeneity of globular domains, compared with greater uniformity of EHS NC1 hexamers. We conclude that EHS NC1 contains an epitope(s) that is reactive with human autoantibodies to hGBM NC1. However, the immune response in Goodpasture's syndrome may involve antibodies directed against epitopes which are present in greater density and on a more complex array of peptides in the hGBM NC1 than in EHS NC1.

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV).

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.