DIFFERENT IMMUNOLOGICAL 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, TBM-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. For tubular NC1, five-fold higher NC1 concentrations were necessary to achieve the same effect. In contrast, for intestinal NC1, more than 30-fold higher protein concentrations of the dissociated NC1 bound only 40% of anti-GBM antibodies. PBM-NC1 did not bind any antibodies, even at these high NC1 concentrations. We conclude, that the differences in the amount of accessible epitopes may possibly explain the preferential involvement of the lungs and the kidneys in the autoimmune Goodpasture's syndrome and that this distribution may be due mainly to a regional expression of the alpha 3(IV) collagen chain.