Crystal Structure of NC1 Domains

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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.

Section: Discussionmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Goodpasture Autoantibodies Unmask Cryptic Epitopes by Selectively Dissociating Autoantigen Complexes Lacking Structural Reinforcement

Borza

Bondar

Colon

et al. 2005

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“…In the GBM, an ␣1⅐␣2(IV) network and a distinct ␣3⅐␣4⅐␣5(IV) network have been identified based on differential solubilization with pseudolysin (7) and analysis of collagenase-solubilized NC1 hexamers (8). The protomer and network organization of ␣1(IV) and ␣2(IV) chains has been well established and confirmed by the recent determination of the crystal structure of the [(␣1) 2 ␣2] 2 (IV) NC1 hexamer (9,10). In contrast, little is known about the organization of ␣3(IV), ␣4(IV), and ␣5(IV) chains, i.e.…”

mentioning

confidence: 79%

Quaternary Organization of the Goodpasture Autoantigen, the α3(IV) Collagen Chain

Borza

Bondar

Todd

et al. 2002

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Goodpasture's (GP) disease is caused by autoantibodies that target the ␣3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (E A and E B ) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the ␣3(IV), ␣4(IV), and ␣5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (␣3) 2 (␣4) 2 (␣5) 2 and to contain cross-linked ␣3-␣5 heterodimers and ␣4-␣4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the ␣3, ␣4, and ␣5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the ␣3, ␣4, and ␣5 chains of one protomer connect with the ␣5, ␣4, and ␣3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the E A region, is sequestered within the ␣3␣4␣5 protomer near the triple-helical junction, at the interface between the ␣3NC1 and ␣5NC1 domains, whereas the E B epitope is sequestered at the interface between the ␣3NC1 and ␣4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the ␣3, ␣4, ␣5, and ␣6 chains in Alport syndrome.

“…However, the accessibility of the non-RGD motifs for ␣ v ␤ 3 integrin within the collagen IV network of basement membrane is still unknown. Homology modeling based on the crystal structure of native ␣1.␣2 NC1 hexamer (44) suggests that non-RGD integrin-binding motifs of the ␣3NC1 domain could be buried within the ␣3⅐␣4⅐␣5 hexamer and therefore not accessible for binding. It should be noted that among the known mammalian sequences, the RGD site proximal to ␣3NC1 domain is unique for the human species.…”

Section: Discussionmentioning

confidence: 99%

αvβ3 and αvβ5 Integrins Bind Both the Proximal RGD Site and Non-RGD Motifs within Noncollagenous (NC1) Domain of the α3 Chain of Type IV Collagen

Pedchenko

Zent

Hudson

2004

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101

103

The NC1 domains of human type IV collagen, in particular ␣3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant ␣3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-␣3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the antiangiogenic activity is attributed exclusively to ␣ v ␤ 3 integrin interactions with non-RGD motifs of the RGD-␣3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for ␣ v ␤ 3 integrin in several proteins. In the present study, we used the ␣3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of ␣ v ␤ 3 and ␣ v ␤ 5 integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the ␣ v ␤ 3 integrin-binding sites of the ␣3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the ␣3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the antiangiogenic activity of the RGD-␣3NC1 domain.Type IV collagen is the major constituent of basement membranes, a specialized form of extracellular matrix underlying all epithelia, that compartmentalizes tissues and provides molecular signals for influencing cell behavior. The type IV collagen family is comprised of six ␣-chains (␣1-␣6) that assemble into three kinds of triple-helical protomers of different chain composition. Each protomer has three functional domains: a 7 S domain at the N terminus, a long triple-helical collagenous domain in the middle of the molecule, and a trimeric noncollagenous (NC1) domain at the C terminus. Protomers selfassemble into networks by end-to-end associations that connect four 7 S domains at one end and connect two NC1 trimeric domains at the other end, forming an NC1 hexamer configuration (1). Three types of networks are known: an ␣1⅐␣1⅐␣2 network, present in the basement membranes of all tissues and animal phyla and ␣3⅐␣4⅐␣5 and ␣1⅐␣2⅐␣5.␣6 networks that have a restricted tissue distribution. These networks are essential for tissue development and function. They provide mechanical stability, a scaffold for assembly of other macromolecular components, and act as a ligand for integrins, receptors that mediate cell adhesion, migration, growth, and differentiation.Cell ...