Quaternary Organization of the Goodpasture Autoantigen, the α3(IV) Collagen Chain (1)
Goodpasture’s (GP) autoantibodies are specifically targeted to the non-collagenous domain (NC1) of the α3(IV) chain, the “Goodpasture autoantigen,” one of the six chains that comprise the collagen IV family. Two conformational GP epitopes, designated EA and EB, have been localized within the α3(IV) NC1 domain at residues 17–31 and 127–141, respectively. In the glomerular basement membrane (GBM) an α1·α2(IV) network and a distinct α3·α4·α5(IV) network have been identified based on differential solubilization with pseudolysin and analysis of collagenase-solubilized NC1 hexamers.
The hexamer population from a preparation of human glomeruli contains all six chains of type IV collagen. The α6-containing hexamers were removed from the mixture by absorption to an anti-α6 affinity column (mAb B66). The B66-bound fraction, representing ∼1% of the total hexamers, contained the α6 NC1 domain along with the α1, α2, and α5 NC1 domains. The unbound hexamers, containing the α1–α5 NC1 domains, were further analyzed with Mab3 (anti-α3), mAb RH45 (anti-α4), and mAb B51 (anti-α5). Mab3 bound hexamers that contained only the α3, α4, and α5 NC1 domains, as described previously, whereas those not bound contained only α1 and α2 NC1 domains. Likewise, mAb RH45 bound hexamers consisting of the α3, α4, and α5 NC1 domains, whereas the unbound hexamers contained only α1 and α2 NC1 domains. mAb B51 bound hexamers containing α3, α4, and α5 NC1 domains, while the unbound fraction contained only α1 and α2 NC1. The α3, α4, and α5 NC1 domains were found in approximately equimolar proportions: 37.4% ± 6.0%, 33.7% ± 6.0%, and 28.9 ± 7.8%, respectively. These results indicate that the hexamer has a composition of (α3)2(α4)2(α5)2.
H11 (to α1 NC1) and H22 (to α2 NC1) mAbs precipitated their respective NC1 monomers and homodimers. Likewise, mAb H43 (to α4 NC1) precipitated only α4 NC1 monomers and α4 NC1 dimers. Several antibodies to the α3 NC1 domain (GPA autoantibodies, mAb EB3, mAb H31) precipitated α3 NC1 monomers and α3 NC1 dimersalong with α5 NC1 dimers of the same size, demonstrating the existence of α3-α5 NC1 heterodimers. Conversely, mAb H52 (to α5 NC1) precipitated α5 NC1 monomers and α5 NC1 dimers along with α3 NC1 dimers, confirming the existence of α3-α5 NC1 heterodimers.
Due to the high sequence homology among the six NC1 domains (52–83% identity), the α3·α4·α5 NC1 hexamer must have a similar tertiary and quaternary structure. The NC1 hexamer is formed of two identical trimeric caps, each derived from one protomer, that interact through a large planar surface. several antibodies to the α3 NC1 domain could selectively block the interaction of α3 NC1 with either α4 or α5 NC1 domains, but not both. Because the epitopes of these antibodies are known and they are located in the proximity of either x or y NC1 domains. Anti-α3 antibodies did not react with immobilized α4 or α5 monomers, indicating that when an α3 NC1-antibody complex bound to the immobilized α4 and/or α5 NC1 domains.
NC1 hexamers have revealed the existence of three distinct collagen IV networks: an ubiquitous one containing the α1 and α2(IV) chains; another containing the α3, α4 and α5(IV) chains, found in the GBM, and other containing the α1, α2, α5, and α6(IV) chains, found in the smooth muscle basement membranes. The organization of chains at the protomer and network level has been determined for the α1·α2(IV) and α1·α2·α5·α6(IV) networks, but not for the α3·α4·α5(IV) network. An α3·α4·α5(IV) network composed of α3α4α5 protomers exists in the GBM. An α1·α2·α5·α6(IV) network composed of (α1)2α2 and (α5)2α6 protomers exists in Bowman’s capsule. An α1·α2(IV) network exists in the GBM, Bowman’s capsule, and mesangial matrix. Within the α3α4α5 protomer, the EA epitope region interfaces with the α5 NC1 domain, and the EB epitope region interfaces with the α4 NC1 domain. Within the EA region of the α3 NC1 domain, four amino acids (Ala-18, Ile-19, Val-27, and Pro-28) were found critical for binding of GPA autoantibodies, and outside this region, Gln-57 may also be required.
