Folding of collagen IV

Abstract

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.

Mammalian collagen IV

“…Pepsin-treated molecules which lacked their NC1 domains were also shown to lack the ability to form network (Yurchenco and Furthmayr, 1984). Using rotary shadowing technique, a time-dependent reassembly of heatdenatured collagen IV molecules was clearly visualized, demonstrating a zipper-like refolding fashion of the triple-helical molecule starting from the C-terminal NC1 domains and proceeding toward the N-terminal ends (Dolz et al, 1988). Direct evidence that the chain specificity of collagen assembly is encoded by the NC1 domains was first demonstrated using purified NC1 hexamers isolated from BMs of different tissues .…”

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

“…As determined by trypsin digestion (presented below), the N-terminal part of the collagen sequence is somewhat unstable. As previously reported, the unfolded single chain collagenous polypeptide cannot be observed with this technique (41). 7 -NC2XIX KnotThe QF constructs were purposely designed to keep the C termini of the collagenous domain of interest in a trimeric prestaggered conformation even at higher temperature when the collagenous domain is unfolded.…”

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

“…First, interactions among NC1 domains initiated assembly of three chains into a triplehelical protomer (26). Second, NC1 domains mediate the association of two protomers head-to-head, forming at the junction an NC1 hexamer, which in turn is stabilized by cross-links (5).…”