BACKGROUND In Goodpasture’s disease, circulating autoantibodies bind to the noncollagenous-1 (NC1) domain of type IV collagen in the glomerular basement membrane (GBM). The specificity and molecular architecture of epitopes of tissue-bound autoantibodies are unknown. Alport’s post-transplantation nephritis, which is mediated by alloantibodies against the GBM, occurs after kidney transplantation in some patients with Alport’s syndrome. We compared the conformations of the antibody epitopes in Goodpasture’s disease and Alport’s post-transplantation nephritis with the intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis. METHODS We used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains. Circulating antibodies were analyzed in 57 patients with Goodpasture’s disease, and kidney-bound antibodies were analyzed in 14 patients with Goodpasture’s disease and 2 patients with Alport’s post-transplantation nephritis. The molecular architecture of key epitope regions was deduced with the use of chimeric molecules and a three-dimensional model of the α345NC1 hexamer. RESULTS In patients with Goodpasture’s disease, both autoantibodies to the α3NC1 monomer and antibodies to the α5NC1 monomer (and fewer to the α4NC1 monomer) were bound in the kidneys and lungs, indicating roles for the α3NC1 and α5NC1 monomers as autoantigens. High antibody titers at diagnosis of anti-GBM disease were associated with ultimate loss of renal function. The antibodies bound to distinct epitopes encompassing region EA in the α5NC1 monomer and regions EA and EB in the α3NC1 monomer, but they did not bind to the native cross-linked α345NC1 hexamer. In contrast, in patients with Alport’s post-transplantation nephritis, alloantibodies bound to the EA region of the α5NC1 subunit in the intact hexamer, and binding decreased on dissociation. CONCLUSIONS The development of Goodpasture’s disease may be considered an autoimmune “conformeropathy” that involves perturbation of the quaternary structure of the α345NC1 hexamer, inducing a pathogenic conformational change in the α3NC1 and α5NC1 subunits, which in turn elicits an autoimmune response. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Type IV collagen, the major component of basement membranes (BMs), is a family of six homologous chains (␣1-␣6) that have a tissue-specific distribution. The chains assemble into supramolecular networks that differ in the chain composition. In this study, a novel network was identified and characterized in the smooth muscle BMs of aorta and bladder. The noncollagenous (NC1) hexamers solubilized by collagenase digestion were fractionated by affinity chromatography using monoclonal antibodies against the ␣5 and ␣6 NC1 domains and then characterized by two-dimensional gel electrophoresis and Western blotting. Both BMs were found to contain a novel ␣1⅐␣2⅐␣5⅐␣6 network besides the classical ␣1⅐␣2 network. The ␣1⅐␣2⅐␣5⅐␣6 network represents a new arrangement in which a protomer (triplehelical isoform) containing the ␣5 and ␣6 chains is linked through NC1-NC1 interactions to an adjoining protomer composed of the ␣1 and ␣2 chains. Re-association studies revealed that the NC1 domains contain recognition sequences sufficient to encode the assembly of both networks. These findings, together with previous ones, indicate that the six chains of type IV collagen are distributed in three major networks (␣1⅐␣2, ␣3⅐␣4⅐␣5, and ␣1⅐␣2⅐␣5⅐␣6) whose chain composition is encoded by the NC1 domains. The existence of the ␣1⅐␣2⅐␣5⅐␣6 network provides a molecular explanation for the concomitant loss of ␣5 and ␣6 chains from the BMs of patients with X-linked Alport's syndrome. The basement membrane (BM),1 a continuous sheet of extracellular matrix, separates epithelial cells from the underlying stroma and plays important roles in normal biological functions (such as cell adhesion, growth, and differentiation; tissue repair; and molecular ultrafiltration) as well as in pathological events (such as cancer cell invasion and metastasis). Moreover, degradation and de novo synthesis of vascular BMs are critical events in the angiogenesis processes. BMs function is impaired in hereditary and acquired diseases in which type IV collagen is affected, including Alport's syndrome, a hereditary form of progressive renal disease; diffuse leiomyomatosis, a benign proliferation of smooth muscle cells; and Goodpasture syndrome, an anti-type IV collagen autoimmune disease (1).Type IV collagen is the major structural component of the BM, and it consists of a family of six homologous ␣(IV) chains, designated ␣1-␣6 (1). Each chain is characterized by a long collagenous domain of ϳ1400 residues of Gly-X-Y repeats, interrupted by ϳ20 short noncollagenous sequences, and by a noncollagenous (NC1) domain of ϳ230 residues at the carboxyl terminus. Three ␣(IV) chains assemble into triple-helical molecules (protomers) that further associate to form supramolecular networks by dimerization at the carboxyl terminus through NC1 domains and by formation of tetramers at the amino terminus (2). The chain composition, and thus the properties of the type IV collagen networks are influenced by two factors. First, the chain composition of networks is limited by chain availability...

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 (␣1 to ␣5) of the six chains of type IV collagen, organized into an ␣1⅐␣2(IV) and an ␣3⅐␣4⅐␣5(IV) network. In Alport syndrome, mutations in any of the genes encoding the ␣3(IV), ␣4(IV), and ␣5(IV) chains cause the absence of the ␣3⅐␣4⅐␣5 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 ␣5 chain was shown to be linked to the ␣3 and ␣4 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 ␣1-␣5(IV) NC1 monomers. The results showed that NC1 monomers associate to form nativelike hexamers characterized by two distinct populations, an ␣1⅐␣2 and ␣3⅐␣4⅐␣5 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 ␣1⅐␣2 and ␣3⅐␣4⅐␣5 networks of GBM. Moreover, hexamer formation from the ␣3, ␣4, and ␣5 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 ␣3⅐␣4⅐␣5 network by interfering with the assembly of the ␣3⅐␣4⅐␣5 NC1 hexamer.

Rapidly progressive glomerulonephritis in Goodpasture disease is mediated by autoantibodies binding to the non-collagenous NC1 domain of ␣3(IV) collagen in the glomerular basement membrane. Goodpasture epitopes in the native autoantigen are cryptic (sequestered) within the NC1 hexamers of the ␣3␣4␣5(IV) collagen network. The biochemical mechanism for crypticity and exposure for autoantibody binding is not known. We now report that crypticity is a feature of the quaternary structure of two distinct subsets of ␣3␣4␣5(IV) NC1 hexamers: autoantibody-reactive M-hexamers containing only monomer subunits and autoantibody-impenetrable D-hexamers composed of both dimer and monomer subunits. Goodpasture antibodies only breach the quaternary structure of M-hexamers, unmasking the cryptic epitopes, whereas Dhexamers are resistant to autoantibodies under native conditions. The epitopes of D-hexamers are structurally sequestered by dimer reinforcement of the quaternary complex, which represents a new molecular solution for conferring immunologic privilege to a potential autoantigen. Dissociation of non-reinforced M-␣3␣4␣5(IV) hexamers by Goodpasture antibodies is a novel mechanism whereby pathogenic autoantibodies gain access to cryptic B cell epitopes. These findings provide fundamental new insights into immune privilege and the molecular mechanisms underlying the pathogenesis of human autoimmune Goodpasture disease.

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the ␣1(IV) and ␣2(IV) NC1 domains. We show that the differential affinity of ␣2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the ␣1(IV) NC1 domain, we conclude that the ␣2(IV) chain plays a regulatory role in directing chain composition in the assembly of (␣1) 2 ␣2 triple-helical molecule. Detailed crystal structure analysis of the [(␣1) 2 ␣2] 2 NC1 hexamer and sequence alignments of the NC1 domains of all six ␣-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a ␤-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.Collagens comprise a major superfamily of extracellular matrix (ECM) 3 proteins that play a key role in the structural integrity of all tissues. At least 27 different collagen types, consisting of 42 distinct gene products, have been identified in vertebrates, underlining their vast diversity in biological functions such as tissue compartmentalization and specialization during the development (1). In the endoplasmic reticulum, the newly synthesized collagen chains assemble into triple-helical molecules with specific chain compositions, which oligomerize to form supramolecular structures, including filaments and networks after secretion to the ECM.Some collagens are obligate homotrimers, such as collagen III, which comprises three identical pro-␣1(III) chains forming an ␣1(III) 3 procollagen, whereas others form heterotrimers containing at least one different ␣-chain; such as collagen I (2). Despite significant sequence identity and a propensity to form triple helices, procollagen chains have an extraordinary ability to discriminate between each other in the endoplasmic reticulum to form specific collagen types. For example, skin fibroblasts express six highly homologous but genetically distinct fibrilforming procollagen chains that are assembled in a type-specific manner to form type I, III, and V collagens. The mechanism by which different collagen chains are selected for assembly is a fundamental question...