Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV)

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.

Section: Triple Helical and Network Organization Of The ␣3(iv)mentioning

confidence: 99%

Section: Organization Of Chains Within the ␣3⅐␣4⅐␣5(iv) Network-mentioning

confidence: 99%

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

Borza

Bondar

Todd

et al. 2002

“…The cross-links connect ␣1-like monomers (␣1-␣1, ␣1-␣5, and ␣3-␣5) and ␣2-like monomers (␣2-␣2, ␣2-␣6, and ␣4 -␣4) (3,4). For two decades, the reduc-ible dimers were thought to consist of monomers bound by disulfide cross-links (5,6). However, the recent x-ray crystal structures of the NC1 hexamers of bovine lens capsule basement membrane and human placenta basement membrane, determined independently by us (7) and Than et al (8), respectively, have disproved this hypothesis.…”

mentioning

confidence: 99%

Identification of S-Hydroxylysyl-methionine as the Covalent Cross-link of the Noncollagenous (NC1) Hexamer of the α1α1α2 Collagen IV Network

Vanacore

Friedman

Ham

et al. 2005

Collagen IV networks are present in all metazoans as components of basement membranes that underlie epithelia. They are assembled by the oligomerization of triple-helical protomers, composed of three ␣-chains. The trimeric noncollagenous domains (NC1) of each protomer interact forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce/cross-link the quaternary structure of hexamer. Recently, the crosslink site of the ␣1␣1␣2 network was identified, on the basis of x-ray crystal structures at 1.9-Å resolution, in which the side chains of Met 93 and Lys 211 were proposed to be connected by a novel thioether bond (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P. Collagen IV networks are components of basement membranes that underlie epithelia, compartmentalize tissue, and influence cell behavior. The networks are assembled from a family of six polypeptide chains (␣1-␣6) that associate forming three subtypes of triple-helical protomers with distinct chain compositions: ␣1␣1␣2, ␣3␣4␣5, and ␣5␣5␣6 (1, 2). The protomers self-assemble by end-to-end associations in which the amino termini of four protomers associate tail-to-tail forming the 7 S domain, and the carboxyl termini of two protomers associate head-to-head through the noncollagenous (NC1) 1 domains, forming dimers. At the interface of the head-to-head connection, the trimeric NC1 domains exist as a hexamer, a stable complex that can be excised by cleavage with collagenase for in vitro studies.The NC1 domain plays a pivotal role in the assembly of the distinct collagen IV networks. In protomer assembly, the NC1 domains (monomers) of three chains interact, forming a NC1 trimer, to select and register chains for triple-helix formation. In the network assembly, the NC1 trimers of two protomers interact, forming a NC1 hexamer structure, to select and connect protomers. Upon exposure to acidic pH or denaturants, isolated NC1 hexamer dissociates into monomers and dimers, the latter reflecting the presence of cross-links that stabilize the trimer-trimer interface. The cross-links connect ␣1-like monomers (␣1-␣1, ␣1-␣5, and ␣3-␣5) and ␣2-like monomers (␣2-␣2, ␣2-␣6, and ␣4 -␣4) (3, 4). For two decades, the reduc- 1 The abbreviations used are: NC1, non-collagenous domain; PBM, placenta basement membrane; GdnHCl, guanidine hydrochloride; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; LC-ESI/MS/MS and LC-ESI/MS 3 , liquid chromatography electrospray tandem mass spectrometry; HPLC, high performance liquid chromatography; DTT, dithiothreitol; Hyl, hydroxylysine; MS 3 , third fragmentation mass spectrometry.

“…Subsequent studies of ␣1.␣2, ␣3.␣4.␣5, and ␣1.␣2-␣5.␣6 collagen IV networks have shown that the cross-links connect ␣1-like monomers (␣1-␣1, ␣1-␣5, and ␣3-␣5) and ␣2-like monomers (␣2-␣2, ␣2-␣6, and ␣4-␣4) (9,10). For two decades, the reducible dimers were thought to be disulfide-linked monomers (7,11). However, the recent x-ray crystal structures of the NC1 hexamers of bovine lens capsule basement membrane (LBM) and human placenta basement membrane (hPBM), determined independently by us (4) and Than et al (6), respectively, have disproved this hypothesis.…”

mentioning

confidence: 99%

The α1.α2 Network of Collagen IV

Vanacore

Shanmugasundararaj

Friedman

et al. 2004

Collagen IV is a major protein component of basement membranes, a specialized form of extracellular matrix underlying epithelia that compartmentalizes tissues and provides molecular signals for influencing cell behavior. The collagen IV family is composed of six ␣-chains (␣1 to ␣6) that assemble into three kinds of triple-helical protomers (1). Each protomer has three functional domains, a 7S domain at the N terminus, a long triple-helical collagenous domain in the middle of the molecule, and a noncollagenous (NC1) 1 domain trimer at the C terminus. Protomers self-assemble into networks by end-to-end associations that connect four molecules at the N terminus, forming a 7S tetramer, and two molecules at the C terminus, forming a NC1 domain hexamer (1). Three types of networks are known: an ␣1.␣2 network present in the basement membranes of all metazoa and an ␣3.␣4.␣5 network and an ␣1.␣2-␣5.␣6 network that have restricted distributions. The networks are essential for tissue function, because they provide mechanical stability, provide a scaffold for assembly of other macromolecules, and serve as ligands for integrins (1-3).The NC1 domain plays a pivotal role in the assembly of distinct networks. The specificity for chain selection is governed by recognition sequences encoded within NC1 domains of the respective six ␣-chains (4, 5). In protomer assembly, the NC1 domains (monomers) of three chains interact, forming an NC1 trimer, to select and register chains for triple-helix formation. In network assembly, the NC1 trimers of two protomers interact, forming an NC1 hexamer structure, to select and connect protomers. This trimer-trimer interface is stabilized by a putative covalent cross-link, which may exist in all three collagen IV networks (6).The existence of cross-links (reducible and nonreducible) was first proposed from studies of the NC1 hexamer isolated by collagenase digestion of ␣1.␣2 collagen IV networks of human placenta, aorta, and mouse tumor (7,8). Upon exposure to acidic pH or denaturants, the NC1 hexamer dissociates into monomers and dimers, the later reflecting the cross-links. Subsequent studies of ␣1.␣2, ␣3.␣4.␣5, and ␣1.␣2-␣5.␣6 collagen IV networks have shown that the cross-links connect ␣1-like monomers (␣1-␣1, ␣1-␣5, and ␣3-␣5) and ␣2-like monomers (␣2-␣2, ␣2-␣6, and ␣4-␣4) (9, 10). For two decades, the reducible dimers were thought to be disulfide-linked monomers (7, 11). However, the recent x-ray crystal structures of the NC1 hexamers of bovine lens capsule basement membrane (LBM) and human placenta basement membrane (hPBM), determined * This work was supported in part by Grants DK18381 (to B. G. H.), DK065123 (to B. G. H.), DK62524 (to M. S.), and RR017806 (to D. B. F) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The atomic coordinates and structure factors (codes 1T60 and...