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Chloride Ion Signals Extracellular Basement Membrane Assembly


By: Carol A. Rouzer, VICB Communications
Published:  June 17, 2016



New studies show how Cl- triggers and stabilizes the interaction between collagen IV protomers during the formation of the basement membrane matrix.


Basement membrane is an important structural component of the extracellular milieu of epithelial, endothelial, muscle, fat, and decidua cells. It defines cellular compartments, provides reinforcement for tissue architecture, guides cell migration and adhesion, delineates cell polarity, and modulates tissue repair and regeneration. Collagen IV, a primary constituent of basement membrane, serves as a scaffold for other proteins, such as growth factors, laminins, and proteoglycans, while also providing tensile strength. Mutation of collagen IV can lead to disorders such as Alport’s syndrome, a debilitating kidney disease, and hemorrhagic stroke. Thus, understanding collagen IV structure and assembly is critical to our knowledge of tissue and organ architecture in both health and disease. This led Vanderbilt Institute of Chemical Biology member Billy Hudson and his laboratory to explore the role of chloride ion in collagen IV assembly [C. F. Cummings, V. Pedchenko, K. L. Brown, et al., J. Cell Biol., 213, 479].


Basement membrane collagen IV is a two-dimensional polymer of alpha chain monomers comprising an N-terminal collagenous domain and a C-terminal noncollagenous (NC1) domain. The first step of collagen IV assembly is formation of the protomer by trimerization of three monomers. During trimerization, the collagenous domains of the monomers intertwine to produce a triple helical tail, and the NC1 domains form a globular head  (Figure 1 A&B). Then, two protomers join head-to-head at the NC1 domains, while four protomers link at the N-terminus of the triple-helical tail (Figure 1 B&C). The association of the NC1 domains of two protomers during collagen IV assembly produces an NC1 hexamer. Stabilization of these hexamers occurs through the formation of sulfilimine cross-links catalyzed by peroxidasin. This enzyme converts Br- to HOBr, the oxidant responsible for joining the sulfur atom of methionine-93 in the NC1 domain of one protomer to the side chain nitrogen atom of hydroxylysine-211 in the NC1 domain of the opposing protomer. Binding sites for Ca2+, Cl- and K+ are visble in the crystal structure of NC1 hexamers (Figure 1E). This latter finding led the Hudson lab to hypothesize that ions may play a role in collagen IV assembly.


(A) Basement membranes are an important structural component of the extracellular matrix of many cell types, including epithelial cells, Schwann cells, and myocytes. (B) A primary building block of basement membranes is collagen IV, which is formed from alpha chain monomers. Trimerization of three of these monomers yields a protomer comprising a long triple helical region and a C-terminal globular noncollagenous domain (NC1). (C & D). The NC1 domains of two protomers dimerize, and the N-terminal regions of the triple helical domains of four hexamers then join to form a network. (E) The NC1 hexamer formed by the dimerization of two protomers is stabilized by sulfilimine cross-links through the action of peroxidasin and Br-. Binding sites for Ca2+, K+, and Cl- are evident in X-ray crystal structures of the NC1 hexamer. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from C. F. Cummings, V. Pedchenko, K. L. Brown, et al., J. Cell Biol., 213, 479. Copyright 2016 C. F. Cummings, V. Pedchenko, K. L. Brown, et al.



The investigators first tested their hypothesis using isolated NC1 hexamers from bovine lens basement membrane (LBM). They found that LBM NC1 hexamers dissociated when they were transferred from NaCl-containing Tris-buffered saline (TBS) to Tris-acetate buffer (TrisAc), which was devoid of NaCl. Uncross-linked NC1 hexamers isolated from PFHR9 cell cultures exhibited a similar dissociation when transferred from TBS to TrisAc. When isolated LBM monomers were transferred back into TBS, they reassociated into hexamers. Hexamer formation occurred readily at concentrations of NaCl normally found in extracellular fluids in vivo (~100 mM). Individual tests of Na+ and Cl- ions indicated that Cl- was the active species. Br- and I- could also induce hexamer formation; however they were not effective at their much lower physiological concentrations (50-100 μM). Although K+ and Ca2+ binding sites are present in collagen IV NC1 domains, these ions had no direct effect on hexamer assembly.


To facilitate the study of collagen IV assembly, the investigators constructed r-Prot, a simplified model of the collagen alpha chain. The r-Prot monomer comprised the NC1 domain and a tail of 28 GXY repeats. The chosen GXY sequences matched those of the α1 or α2 chains, and the length was sufficient to allow stable triple helix formation. The researchers incorporated an α1β2 integrin binding site into the GXY repeat region of the protein to mimic an important function of collagen IV in basement membrane (Figure 2A). Incubation of r-Prot monomers at a 2:1 α1:α2 ratio in TBS led to the formation of both protomers and protomer dimers as indicated by the appearance of new peaks on size-exclusion chromatography (Figure 2B). Collagenase treatment of protomers, which digests the collagenous domains of collagen alpha chains, yielded monomeric NC1 domains, whereas treatment of the protomer dimers yielded NC1 hexamers. These findings indicated that the triple helical tail is required to stabilize NC1 trimers, but once NC1 hexamers have formed, the tail is no longer needed. Both the protomers and dimers could bind to the α2 integrin I-domain or to integrin-expressing HT1080 cells, confirming that r-Prot protomers and dimers retain characteristic functions of collagen IV assemblies.


FIGURE 2. (A) Construction of the r-Prot model for collagen IV assembly. The monomers comprise the NC1 domain fused to 28 GXY repeats, which provide a tail long enough to form a triple helix. GXY sequences were chosen to match those of the α1 and α2 chains of collagen IV. The NC1 domain contains the Cl- binding sites observed in the structure of the native protein. (B) An α2β1 integrin binding site (red) was incorporated into the triple helical segment, and binding of the α2 I-domain of integrin (magenta) to this site was confirmed. (C) The α1 and α2 monomers eluted similarly at about 14 mL from a size exclusion column. (D) Incubation with Tris-buffered saline containing Cl- produced two new peaks on size exclusion chromatography. These correspond to the trimeric protomer (11 min, P) and the dimerized protomer (9 min, P2). Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from C. F. Cummings, V. Pedchenko, K. L. Brown, et al., J. Cell Biol., 213, 479. Copyright 2016 C. F. Cummings, V. Pedchenko, K. L. Brown, et al.



The investigators found that incubation of r-Prot protomer dimers in TrisAc resulted in their dissociation to protomers. Exposure of protomers to heat led to further dissociation to monomers. Cooling of the monomers in the absence of Cl- resulted in their reassembly to protomers; however, formation of protomer dimers required addition of Cl-. Incubation of fully assembled protomer dimers with peroxidasin and Br- led to NC1 cross-linking and stabilized the proteins to dissociation in TrisAc. These results suggested that protomer formation can occur in the absence of Cl-, but Cl- is required for the association of the NC1 domains of two protomers to form a hexameric structure.

The researchers combined molecular modeling, molecular dynamics, and X-ray crystallography to explore the mechanism by which Cl- induces protomer dimerization. They discovered that the Cl- binding site in the NC1 hexamer comprises alanine-74, serine-75, arginine-76, glutamine-77, and aspartate-78. The Cl- coordinates the backbone amide groups of these amino acids. In the absence of Cl-, arginine-76 forms salt bridges with aspartate-78 and glutamate-40, helping to stabilize the NC1 domain in the protomer. Binding of Cl- disrupts these salt bridges and alters the conformation of arginine-76. As a result, its side-chain projects outward so that it can interact with the NC1 domain of an approaching protomer. As dimerization occurs, arginine-76 forms new salt bridges with glutamate-175 and arginine-179 of the opposing protomer, stabilizing the dimer interface (Figure 3). The investigators confirmed the importance of these salt bridges by expressing recombinant α1 and α2 chains bearing an R76A mutation. These monomers were able to form protomers but not protomer dimers.



FIGURE 3. Effect of Cl- on collagen IV assembly. (A) in the absence of Cl-, R76 of the NC1 domain forms ionic interactions with E40 and D78, stabilizing the local structure of the NC1 domain in the protomer. (B) Addition of Cl- disrupts these ionic interactions. (C) Cl- interacts with the backbone of R76, restricting its conformation, and directing it outward so it can interact with R179 and E175 of an opposing NC1 domain as the two protomers dimerize. (D) The resulting salt bridges stabilize the interactions between the two protomers. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from C. F. Cummings, V. Pedchenko, K. L. Brown, et al., J. Cell Biol., 213, 479. Copyright 2016 C. F. Cummings, V. Pedchenko, K. L. Brown, et al.



To explore the importance of Cl- to basement membrane formation in intact cells, the investigators cultured PFHR9 cells in the presence of medium containing a low Cl- concentration. They observed that the wet weight and sulfilimine cross-link content of basement membrane produced by the cells under these conditions were less than those obtained from cultures grown in standard medium. However, when they incubated this material in medium containing Cl- followed by HOBr, a fully assembled and cross-linked collagen IV matrix resulted. Further experiments demonstrated that cells grown in low Cl- medium produced poorly organized basement membrane, characterized by aberrant localization of associated proteins such as laminin and peroxidasin. Transfer of the cells to conventional medium resulted in their production of new, correctly organized basement membrane.


To investigate the role of Cl- during basement membrane formation in an intact organism, the investigators developed a model in the fruit fly Drosophila melanogaster. They created flies that expressed mutant α1 collagen transgenes fused to green fluorescent protein (GFP) along with the wild-type α1 protein. The GFP tag enabled them to visualize the expressed proteins in transgenic fly larvae. The researchers discovered that an α1 protein carrying a deletion of the NC1 domain was expressed, but they could find it only in the circulation, indicating that this protein could not be incorporated into a basement membrane assembly. In contrast, a transgenic protein carrying an R76A single amino acid mutation was both expressed and incorporated into basement membrane. These findings confirm that an intact NC1 domain is required for protomer formation, hence the deletion mutation excluded basement membrane incorporation. In contrast, the ability of the R76A mutant to form protomers enabled it to trimerize with wild-type alpha chains, and thereby be incorporated into complete basement membrane matrix.


Together the results suggest a mechanism of basement membrane collagen IV assembly (Figure 4). The investigators propose that monomer synthesis and protomer formation occur intracellularly where Cl- concentration is low. Following secretion into the extracellular space, Cl- promotes dimerization of protomers. This is followed by peroxidasin-mediated cross-linking and tail-to-tail association to yield the two-dimensional matrix. The findings also help to explain diseases, such as Alport’s syndrome, in which mutations in the NC1 domain result in the loss of the collagen IV network in the glomerulus of the kidney leading ultimately to renal failure, requiring dialysis or transplant.



FIGURE 4. Proposed mechanism of collage IV assembly. (A) Alpha chain monomers are produced within the cell and trimerize intracellularly to form the protomers. Cl- concentration inside of the cell is too low to promote dimerization. (B) The protomers are secreted into the extracellular space where Cl- concentration is much higher. Cl- promotes dimerization of the protomers, and then peroxidasin (PXDN) catalyzes sulfilimine cross-link formation. (D) The N-terminal domains of the dimerized protomers then link to form the two-dimensional basement membrane network, which serves as a scaffold for multiple components of the extracellular matrix. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from C. F. Cummings, V. Pedchenko, K. L. Brown, et al., J. Cell Biol., 213, 479. Copyright 2016 C. F. Cummings, V. Pedchenko, K. L. Brown, et al.




View J. Cell Biol. article: Extracellular chloride signals collagen IV network assembly during basement membrane formation








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