Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in ␣ 3(IV), ␣ 4(IV), and ␣ 5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of ␣ 1(IV) and ␣ 2(IV) isoforms because they fail to developmentally switch their ␣ -chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich ␣ 3(IV), ␣ 4(IV), and ␣ 5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate. ( J. Clin. Invest. 1997. 99:2470-2478.)

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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...

Type IV collagen, which is present in all metazoan, exists as a family of six homologous ␣(IV) chains, ␣1-␣ 6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(␣1) 2 .␣2] 2 NC1 hexamer of bovine lens capsule basement membrane at 2.0 Å resolution. The NC1 monomer folds into a novel tertiary structure. The (␣1) 2 .␣2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.