M C

Figure 3 Representation of a hypothetical collagen IV network in which the six characterized u (IV) chains are assembled into three types of collagen IV molecules ((a1)2a2; (<i3)2a4; (a5)2a6) and interactions through the NC1 domains are restricted to homologous molecules. There is evidence for the existence of (a1)2«2 and («3)2a4 but not for («5)2«6. Other collagen IV compositions and interactions expected to occur are not represented in the drawing for simplicity. Bacterial collagenase digestion of the collagen IV network releases the hexameric NC1 domain that yields monomer and related dimers upon dissociation The antigenic determinants recognized by the GP antibodies are hidden within the isolated hexamer and only exist in the «3(IV)NC1-containing material. At the lower right side, the hexamer was analyzed by denaturing electrophoresis and transferred to a nitrocellulose membrane. The membrane was sliced in two and the individual pieces blotted with Goodpasture serum (left) or with monoclonal antibodies recognizing the N-terminal region of the human a3(IV)NC1 (right). The undistinguishable reactive patterns in both monomer (M) and dinner (D) regions localizes the Goodpasture antigen to the u3(IV)NC1 domain.

lated hexamer since the unfolding of the hexamer is required for full GP antibody recognition (Figure 3).

The expression of each individual chain varies greatly among tissues with a more restricted distribution for the a3(IV)-a6(IV) chains. In the glomeruli, the ct3(IV)-a5(IV) chains are coordinately expressed and it appears that these three chains assemble into a collagen IV network different but connected to the more classic and ubiquitous one composed of the al(IV)-a2(IV) chains. Typically in the Alport syndrome, a condition arising from mutations in the a3(IV), c*4(IV) or a5(IV) genes, the glomerulus does not express the a3(IV)-a5(IV) chains. In Alport patients, the glomerular basement membrane displays a split structural appearance caused by the defective fusion of the endothelial and epithelial basement membranes, resulting in a severe impairment of the plasma filtration. This pathology reveals that the a3(IV)-a5(IV) network cements the double-width basement membrane and is a critical structural requirement for the glomerular filtration. This structural-functional relationship is further supported by the renal failure that results from the particular binding of antibodies to the C-terminal domain of a3(IV) in GP patients.

Collagenase digestion of the human a3(IV) chain releases the GP antigen, a 244-residue polypeptide consisting of a short nondigested collagenous sequence at the amino end followed by the NCI domain (Figure 4). The molecular cloning of the different human a(IV) chains and that of a3(IV) from other species has allowed comparative studies that have identified the pathogenic epitope and certain biological features unique to the human antigen. The GP epitope is discontinous, disulfide-dependent and involves at least both ends of the antigen (Figure 4). In humans, the amino-side of the GP epitope is phos-phorylated by type A protein kinases and antigen-

related products produced by alternative exon splicing regulate the process. These alternative products share the phosphorylatable amino-side of the epitope with the primary antigen and diverge from it at the C-terminal region. The shorter, divergent C-terminal regions do not contain the structural requirements to build a classic NCI domain and therefore are not expected to guide triple helix formation. However, they do confer a distinct susceptibility to phosphorylation to the common N-terminal region.

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